Sequestration of carbon dioxide with hydrogen to useful products

ABSTRACT

Provided herein are genetically engineered microbes that include at least a portion of a carbon fixation pathway, and in one embodiment, use molecular hydrogen to drive carbon dioxide fixation. In one embodiment, the genetically engineered microbe is modified to convert acetyl CoA, molecular hydrogen, and carbon dioxide to 3-hydroxypropionate, 4-hydroxybutyrate, acetyl CoA, or the combination thereof at levels greater than a control microbe. Other products may also be produced. Also provided herein are cell free compositions that convert acetyl CoA, molecular hydrogen, and carbon dioxide to 3-hydroxypropionate, 4-hydroxybutyrate, acetyl CoA, or the combination thereof. Also provided herein are methods of using the genetically engineered microbes and the cell free compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 14/426,290, filed Mar. 5, 2015, which is the §371U.S. National Stage of International Application No. PCT/US13/58593,filed Sep. 6, 2013, which claims the benefit of U.S. ProvisionalApplication Ser. No. 61/697,654, filed Sep. 6, 2012, the disclosures ofwhich are incorporated by reference herein in their entireties.

GOVERNMENT FUNDING

The present invention was made with government support under Grant No.DE-AR0000081, awarded by the Department of Energy. The government hascertain rights in this invention.

SEQUENCE LISTING

This application contains a Sequence Listing electronically submittedvia EFS-Web to the United States Patent and Trademark Office as an ASCIItext file entitled “235_01670102_SequenceList_ST25.txt” having a size of1,255 kilobytes and created on Mar. 27, 2017. The information containedin the Sequence Listing is incorporated by reference herein.

BACKGROUND

Carbon dioxide is chemically stable and unreactive, and must be reducedto enable its incorporation into biological molecules. Autotrophicmicroorganisms are able to utilize carbon dioxide as their sole carbonsource and a variety of pathways are known to activate and incorporateit into biomolecules essential for growth and replication. Recently,carbon dioxide fixation pathways have received interest forbiotechnological applications, since this could provide biologicalroutes for de novo generation of fuels and small organic molecules(Hawkins et al., 2011, ACS Catal. 1, 1043-1050).

There are currently at least six natural pathways for the incorporationof inorganic carbon dioxide into cellular carbon (Berg 2011, Appl.Environ. Microbiol. 77, 1925-1936; Berg et al., 2010, Nat. Rev.Microbiol. 8, 447-460). The most recently discovered of these are foundexclusively in extremely thermophilic archaea: the3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) carbon fixation cycle,which operates in members of the crenarchaeal order Sulfolobales ((Berg2011, Appl. Environ. Microbiol. 77, 1925-1936; Berg et al., 2007,Science 318, 1782-1786; Alber et al., 2008, J. Bacteriol. 190,1383-1389; Hugler et al., (2003) Arch. Microbiol. 179, 160-173), and thedicarboxylate/4-hydroxybutyrate (DC/4HB) cycle, which is used byanaerobic members of the orders Thermoproteales and Desulfurococcales(Berg et al., 2007, Science 318, 1782-1786; Huber et al., (2008) PNASUSA 105, 7851-7856). In both cycles, two carbon dioxide molecules areadded to acetyl-CoA (C2) to produce succinyl-CoA (C4), which issubsequently rearranged to acetoacetyl-CoA and cleaved into twomolecules of acetyl-CoA. These pathways differ primarily in regards totheir tolerance to oxygen and the co-factors used for reducingequivalents—NAD(P)H for the 3HP/4HB cycle and ferredoxin/NAD(P)H for theDC/4HB cycle (Berg et al., 2010, Nat. Rev. Microbiol. 8, 447-460;Auernik and Kelly, 2010, Appl. Environ. Microbiol. 76, 931-935). The twoarchaeal pathways also differ in how they link the C02 fixation cycle tocentral metabolism. In the DC/4HB pathway, pyruvate is synthesizeddirectly from acetyl-CoA using pyruvate synthase. In the 3HP/4HBpathway, another half-turn is required to make succinyl-CoA, which isthen oxidized via succinate to pyruvate (Berg 2011, Appl. Environ.Microbiol. 77, 1925-1936; Ramos-Vera et. al., 2011, J. Bacteriol. 193,1201-1211; Estelmann et al., (2011) J. Bacteriol. 193, 1191-1200).

There are 13 enzymes proposed to catalyze the 16 reactions in the3HP/4HB pathway. The first three enzymes convert acetyl-CoA (C2) to 3HP(C3) via an ATP-dependent carboxylation step. Next, 3HP is converted andreduced to propionyl-CoA, carboxylated a second time and rearranged tomake succinyl-CoA (C4). Succinyl-CoA is reduced to 4HB, which isconverted to two molecules of acetyl-CoA in the final reactions of thecycle. Flux analysis and labeling studies have confirmed the operationof this pathway in M. sedula (Berg et al., 2007, Science 318, 1782-1786;Estelmann et al., (2011) J. Bacteriol. 193, 1191-1200).

SUMMARY OF THE INVENTION

Enzymes of the first portion of the3-hydroxypropionate/4-hydroxybutyrate carbon fixation cycle up to theformation of 4-hydroxybutyrate (4HB) have been identified andcharacterized biochemically in their native or recombinant form, mostlyfrom the extremely thermoacidophilic archaeon Metallosphaera sedula(T=70° C., pH=2.0) (See Table 1). The enzymes involved in the conversionof 4HB to two molecules of acetyl-CoA have not been characterized to thesame extent (FIG. 1, E10−E13). Activities corresponding to4-hydroxybutyryl-CoA dehydratase and acetoacetyl-CoA β-ketothiolase havebeen detected in cell extracts, although neither enzyme has beenpurified in its native form or recombinantly produced. Identification ofcandidates for both of these enzymes has been made based on genomeannotation and transcriptomic analysis of autotrophic growth compared toheterotrophy (Auernik and Kelly, 2010, Appl. Environ. Microbiol. 76,931-935; Ramos-Vera et. al., 2011, J. Bacteriol. 193, 1201-1211). Whileneither of the candidate genes for these enzymes has so far beenconfirmed biochemically, their identity is not in dispute because ofstrong homology to known versions in less thermophilic organisms. Thecorresponding gene products in M. sedula are Msed_1321 for the 4HB-CoAdehydratase and Msed_0656 for the acetoacetyl-CoA β-ketothiolase.Further, the polypeptides of the 3-hydroxypropionate/4-hydroxybutyratecarbon fixation cycle have not been genetically engineered forexpression in any system that allows one to take advantage of thestability of the polypeptides at high temperatures.

TABLE 1 Enzymes in the 3HP/4HB Cycle in Metallosphaera sedula EnzymeReference # ORF Enzyme Lit. Ref E1α Msed_0147 acetyl-CoA/propionyl-CoANCE (1, 2) E1β Msed_0148 carboxylase E1γ Msed_1375 E2 Msed_0709malonyl-CoA/succinyl-CoA R (3) reductase E3 Msed_1993 malonatesemialdehyde R (3) reductase E4 Msed_1456 3-hydroxypropionate:CoA ligaseNP (5) E5 Msed_2001 3-hydroxypropionyl-CoA NP, R (4) dehydratase E6Msed_1426 acryloyl-CoA reductase NP (4) E7 Msed_0639 methylmalonyl-CoAepimerase R (6) E8α Msed_0638 methylmalonyl-CoA mutase R (6) E8βMsed_2055 E9 Msed_1424 succinate semialdehyde NP, R (3) reductase E10Msed_0394 4-hydroxybutyrate:CoA ligase R, Msed_0406 Example 1 E11Msed_1321 4-hydroxybutyrl-CoA NCE (7) R, dehydratase Example 8 E12Msed_0399 crotonyl-CoA hydratase/(S)-3- R (8) hydroxybutyrl-CoAdehydrogenase E13 Msed_0656 acetoacetyl-CoA β-ketothiolase NCE (7); R,Example 8 (1) Hügler et al., 2003, Eur. J. Biochem. 270, 736-744; (2)Menendez et al., 1999 J. Bacteriol. 181, 1088-1098; (3) Kockelkorn andFuchs, 2009, J. Bacteriol. 191, 6352-6362; (4) Teufel et al., 2009, J.Bacteriol. 191, 4572-4581; (5) Alber et al., 2008, J. Bacteriol. 190,1383-1389; (6) Han et al., 2012, Appl. Environ. Microbiol. 78:6194-62027; (7) Berg et al., 2007, Science 318, 1782-1786; and (8)Ramos-Vera et. al.., 2011, J. Bacteriol. 193, 1201-1211.

The identity of the crotonyl-CoA hydratase and the(S)-3-hydroxybutyryl-CoA dehydrogenase was recently confirmed, when itwas discovered that both reactions were catalyzed by a singlebifunctional fusion protein (Ramos-Vera et. al., 2011, J. Bacteriol.193, 1201-1211). In the same work, Ramos-Vera et al. tested threedifferent candidates for the 4HB-CoA synthetase, but all failed to showactivity on 4HB. In fact, the primary candidate suggested by theautotrophic transcriptome analysis (Msed_1422) showed no enzymaticactivity on short-chain linear unsubstituted orhydroxy-acids—specifically acetate, propionate, 3HP, 3-hydroxybutyrate,4HB and crotonate. Two other candidates were selected, based on homologyto 4HB-CoA synthetase from T. neutrophilus (Tneu_0420) and 3HP-CoAsynthetase from M. sedula: Msed_1353 and Msed_1291 were recombinantlyproduced and tested for ligase activity. Msed_1353 was active onpropionate and acetate, but not on 4HB. Furthermore, Msed_1291 had noactivity on any of the previously mentioned organic acids. Thus,although cycle function has been confirmed by metabolic flux analysis,and while 4HB-CoA synthetase activity has been measured in cell extractsof autotrophically-grown M. sedula, the enzyme responsible for ligationof CoA to 4HB remains unclear.

In order to identify the missing link in the 3HP/4HB cycle, new methodsfor semi-continuous cultivation of M. sedula in a gas-intensivefermentation system were developed to tease out differentialtranscriptional response of autotrophy-related genes. Strict carbondioxide limitation was used to drive increased operational efficiency ofthe CO₂ fixation enzymes, which hypothetically would increasetranscriptional levels of genes encoding key enzymes to maximize carbonincorporation. Using these conditions for transcriptional analysis, amuch clearer picture emerged concerning the global regulatory changes inM. sedula, as its cellular metabolism switches from autotrophy toheterotrophy. This strategy produced new leads for the genes andcorresponding enzymes responsible for the 4HB-CoA ligation step. Theenzymes were recombinantly produced and shown to catalyze the ligationof CoA to 4HB.

Accordingly, provided herein are genetically engineered microbes. In oneembodiment, the genetically engineered microbe is modified to convertacetyl CoA, molecular hydrogen, and carbon dioxide to3-hydroxypropionate. The 3-hydroxypropionate is produced at increasedlevels compared to a control microbe. In one embodiment, the geneticallyengineered microbe that includes (a) enzymes to fix CO₂ and produce3-hydroxypropionate and (b) an NADPH-dependent hydrogenase. Thegenetically engineered microbe has greater production of3-hydroxypropionate than a control microbe that does not include either(a) or (b). The 3-hydroxypropionate may be produced in the absence oflight energy.

In one embodiment, the genetically engineered microbe includes anexogenous coding region encoding a polypeptide, wherein the polypeptidehas an activity selected from acetyl/propionyl-CoA carboxylase activity,malonyl/succinyl-CoA reductase activity, and malonate semialdehydereductase activity. In one embodiment, the genetically engineeredmicrobe includes an exogenous coding region encoding a polypeptidehaving acetyl/propionyl-CoA carboxylase activity, an exogenous codingregion encoding a polypeptide having malonyl/succinyl-CoA reductaseactivity, and an exogenous coding region encoding a polypeptide havingmalonate semialdehyde reductase activity.

In one embodiment, the genetically engineered microbe is modified toconvert acetyl CoA, molecular hydrogan and carbon dioxide to4-hydroxybutyrate. The 4-hydroxybutyrate is produced at increased levelscompared to a control microbe. In one embodiment, the geneticallyengineered microbe includes (a) enzymes to fix CO₂ and produce4-hydroxybutyrate and (b) an NADPH-dependent hydrogenase. Thegenetically engineered microbe has greater production of3-hydroxypropionate than a control microbe that does not include either(a) or (b). The 4-hydroxybutyrate may be produced in the absence oflight energy.

In one embodiment, the genetically engineered microbe produces3-hydroxypropionate, and the microbe includes an exogenous coding regionencoding a polypeptide, wherein the polypeptide has an activity selectedfrom 3-hydroxypropionate:CoA ligase activity, 3-hydroxypropionyl-CoAdehydratase activity, acryloyl-CoA reductase activity, methylmalonyl-CoAepimerase activity, methylmalonyl-CoA mutase activity, and succinatesemialdehyde reductase activity. In one embodiment, the geneticallyengineered microbe produces 3-hydroxypropionate, and the microbeincludes an exogenous coding region encoding a polypeptide having3-hydroxypropionate:CoA ligase activity, an exogenous coding regionencoding a polypeptide having 3-hydroxypropionyl-CoA dehydrataseactivity, an exogenous coding region encoding a polypeptide havingacryloyl-CoA reductase activity, an exogenous coding region encoding apolypeptide having methylmalonyl-CoA epimerase activity, an exogenouscoding region encoding a polypeptide having methylmalonyl-CoA mutaseactivity, and an exogenous coding region encoding a polypeptide havingsuccinate semialdehyde reductase activity.

In one embodiment, the genetically engineered microbe is modified toproduce acetyl CoA at increased levels compared to a control microbe. Inone embodiment, the genetically engineered microbe is modified toconsume one acetyl CoA molecule, molecular hydrogen and carbon dioxideto produce two acetyl CoA molecules at increased levels compared to acontrol microbe. The acetyl CoA may be produced in the absence of lightenergy.

In one embodiment, the genetically engineered microbe produces4-hydroxybutyrate, and the microbe includes an exogenous coding regionencoding a polypeptide, wherein the polypeptide has an activity selectedfrom 4-hydroxybutyrate:CoA ligase activity, 4-hydroxybutyrl-CoAdehydratase activity, crotonyl-CoA hydratase/(S)-3-hydroxybutyrl-CoAdehydrogenase activity, and acetoacetyl-CoA β-ketothiolase activity. Inone embodiment, the genetically engineered microbe produces4-hydroxybutyrate, and the microbe includes an exogenous coding regionencoding a polypeptide having 4-hydroxybutyrate:CoA ligase activity, anexogenous coding region encoding a polypeptide having4-hydroxybutyrl-CoA dehydratase activity, an exogenous coding regionencoding a polypeptide having crotonyl-CoAhydratase/(S)-3-hydroxybutyrl-CoA dehydrogenase activity, and anexogenous coding region encoding a polypeptide having acetoacetyl-CoAβ-ketothiolase activity.

In one embodiment, the genetically engineered microbe is anextremophile, such as a hyperthermophile. In one embodiment, thehyperthermophile is an archeon. In one embodiment, the archeon is amember of the Order Thermococcales, a member of the Order Sulfolobales,or a member of the Order Thermotogales. In one embodiment, the archeonis Thermococcus kodakarensis, T. onnurineus, Sulfolobus solfataricus, S.islandicus, S. acidocaldarius, or Pyrococcus furiosus.

In one embodiment, an exogenous coding region is operably linked to atemperature sensitive promoter, to a constitutive promoter, or to anon-regulated promoter. In one embodiment, the genetically engineeredmicrobe further includes a hydrogenase, such as a NADPH-dependenthydrogenase. In one embodiment, the genetically engineered microbeincludes exogenous coding regions encoding subunits of theNADPH-dependent hydrogenase. In one embodiment, the subunits of theNADPH-dependent hydrogenase include a hydrogenase alpha subunit and ahydrogenase delta subunit. In one embodiment, the subunits of theNADPH-dependent hydrogenase further include a hydrogenase beta subunitand a hydrogenase gamma subunit.

Also provided herein are methods for using the genetically engineeredmicrobes. In one embodiment, the method includes incubating thegenetically engineered microbe under anaerobic conditions suitable forconverting acetyl CoA, molecular hydrogen, and carbon dioxide to3-hydroxypropionate, to 4-hydroxybutyrate, acetyl CoA, or a combinationthereof. In one embodiment, the method further includes converting the3-hydroxypropionate, 4-hydroxybutyrate, or acetyl CoA into anotherproduct, such as pyruvate or succinate. In one embodiment, the methodfurther includes recovering the 3-hydroxypropionate, 4-hydroxybutyrate,acetyl CoA or other product.

Also provided herein are cell free compositions. In one embodiment, thecell free composition converts acetyl CoA, molecular hydrogen and carbondioxide to 3-hydroxypropionate. The cell free composition includes apolypeptide having acetyl/propionyl-CoA carboxylase activity, apolypeptide having malonyl/succinyl-CoA reductase activity, apolypeptide having malonate semialdehyde reductase activity, and apolypeptide having NADPH-dependent hydrogenase activity. In oneembodiment, the cell free composition converts 3 hydroxypropionate to4-hydroxybutyrate. In such an embodiment, the composition furtherincludes a polypeptide having 3-hydroxypropionate:CoA ligase activity, apolypeptide having 3-hydroxypropionyl-CoA dehydratase activity, apolypeptide having acryloyl-CoA reductase activity, a polypeptide havingmethylmalonyl-CoA epimerase activity, a polypeptide havingmethylmalonyl-CoA mutase activity, and a polypeptide having succinatesemialdehyde reductase activity. In one embodiment, the cell freecomposition converts 4-hydroxybutyrate to acetyl CoA. In such anembodiment, the composition further includes a polypeptide having4-hydroxybutyrate:CoA ligase activity, a polypeptide having4-hydroxybutyrl-CoA dehydratase activity, a polypeptide havingcrotonyl-CoA hydratase/(S)-3-hydroxybutyrl-CoA dehydrogenase activity,and a polypeptide having acetoacetyl-CoA β-ketothiolase activity.

Also provided herein are methods for using a cell free composition. Inone embodiment, the cell free method fixes CO₂, and includes incubatingthe cell free composition under anaerobic conditions suitable for thefixation of CO₂ by the conversion of acetyl CoA, molecular hydrogen andcarbon dioxide to 3-hydroxypropionate. In one embodiment, the methodfurther includes isolating the 3-hydroxypropionate. In one embodiment,the cell free method fixes CO₂, and includes incubating the cell freecomposition under anaerobic conditions suitable for the fixation of CO₂by the conversion of acetyl CoA, molecular hydrogen and carbon dioxideto 4-hydroxybutyrate. In one embodiment, the method further includesisolating the 4-hydroxybutyrate. In one embodiment, the method fixes CO₂and includes incubating the cell free composition under anaerobicconditions suitable for the fixation of CO₂ by the conversion of4-hydroxybutyrate, molecular hydrogen and carbon dioxide to acetyl CoA.In one embodiment, the method further includes isolating the acetyl CoA.In one embodiment, the conditions include a temperature between 60° C.and 80° C.

As used herein, the term “polypeptide” refers broadly to a polymer oftwo or more amino acids joined together by peptide bonds. The term“polypeptide” also includes molecules which contain more than onepolypeptide joined by a disulfide bond, ionic bonds, or hydrophobicinteractions, or complexes of polypeptides that are joined together,covalently or noncovalently, as multimers (e.g., dimers, trimers,tetramers). A polypeptide also may possess non-protein (non-amino acid)ligands including, but not limited to, inorganic iron (Fe), nickel (Ni),inorganic iron-sulfur centers such as [4Fe-4S] clusters, and otherorganic ligands such as carbon monoxide (CO), cyanide (CN) and flavin.Thus, the terms peptide, oligopeptide, enzyme, subunit, and protein areall included within the definition of polypeptide and these terms areused interchangeably. It should be understood that these terms do notconnote a specific length of a polymer of amino acids, nor are theyintended to imply or distinguish whether the polypeptide is producedusing recombinant techniques, chemical or enzymatic synthesis, or isnaturally occurring.

As used herein, “heterologous amino acid sequence” refers to amino acidsequences that are not normally present as part of a polypeptide presentin a wild-type cell. For instance, “heterologous amino acid sequence”includes extra amino acids at the amino terminal end or carboxy terminalof a polypeptide that are not normally part of a polypeptide that ispresent in a wild-type cell.

As used herein, “hydrogenase activity” refers to the ability of apolypeptide(s) to catalyze the formation of reductants such as NADPHfrom molecular hydrogen (H₂), and also refers to the ability to catalyzethe reverse reaction.

As used herein, “identity” refers to structural similarity between twopolypeptides or two polynucleotides. The structural similarity betweentwo polypeptides is determined by aligning the residues of the twopolypeptides (e.g., a candidate amino acid sequence and a referenceamino acid sequence) to optimize the number of identical amino acidsalong the lengths of their sequences; gaps in either or both sequencesare permitted in making the alignment in order to optimize the number ofshared amino acids, although the amino acids in each sequence mustnonetheless remain in their proper order. The structural similarity istypically at least 80% identity, at least 81% identity, at least 82%identity, at least 83% identity, at least 84% identity, at least 85%identity, at least 86% identity, at least 87% identity, at least 88%identity, at least 89% identity, at least 90% identity, at least 91%identity, at least 92% identity, at least 93% identity, at least 94%identity, at least 95% identity, at least 96% identity, at least 97%identity, at least 98% identity, or at least 99% identity. A candidateamino acid sequence can be isolated from a microbe, such as a Pyrococcusspp., including P. furiosus, or a Metallosphaera spp., including M.sedula or can be produced using recombinant techniques, or chemically orenzymatically synthesized. Structural similarity may be determined, forexample, using sequence techniques such as the BESTFIT algorithm in theGCG package (Madison Wis.), or the Blastp program of the blastpsuite-2sequences search algorithm, as described by Tatiana et al., (FEMSMicrobiol Lett, 174, 247-250 (1999)), and available on the NationalCenter for Biotechnology Information (NCBI) website. The default valuesfor all blastp suite-2sequences search parameters may be used, includinggeneral paramters: expect threshold=10, word size=3, short queries=on;scoring parameters: matrix=BLOSUM62, gap costs=existence: 11 extension:1, compositional adjustments=conditional compositional score matrixadjustment. Alternatively, polypeptides may be compared using theBESTFIT algorithm in the GCG package (version 10.2, Madison Wis.). Inthe comparison of two amino acid sequences using the BLAST searchalgorithm, structural similarity is referred to as “identities.”

As used herein, an “isolated” substance is one that has been removedfrom its natural environment, produced using recombinant techniques, orchemically or enzymatically synthesized. For instance, a polypeptide ora polynucleotide described herein can be isolated. With respect to aproduct produced using a method described herein, “isolated” refers toremoval of the product from the medium in which it was produced by agenetically engineered microbe. Preferably, a substance is purified,i.e., is at least 60% free, preferably at least 75% free, and mostpreferably at least 90% free from other components with which it isnaturally associated, or from other components present in the medium inwhich it was produced.

As used herein, the term “polynucleotide” refers to a polymeric form ofnucleotides of any length, either ribonucleotides or deoxynucleotides,and includes both double- and single-stranded RNA and DNA. Apolynucleotide can be obtained directly from a natural source, or can beprepared with the aid of recombinant, enzymatic, or chemical techniques.A polynucleotide can be linear or circular in topology. A polynucleotidemay be, for example, a portion of a vector, such as an expression orcloning vector, or a fragment. A polynucleotide may include nucleotidesequences having different functions, including, for instance, codingregions, and non-coding regions such as regulatory regions.

As used herein, the terms “coding region,” “coding sequence,” and “openreading frame” are used interchangeably and refer to a nucleotidesequence that encodes a polypeptide and, when placed under the controlof appropriate regulatory sequences expresses the encoded polypeptide.The boundaries of a coding region are generally determined by atranslation start codon at its 5′ end and a translation stop codon atits 3′ end. A “regulatory sequence” is a nucleotide sequence thatregulates expression of a coding sequence to which it is operablylinked. Non-limiting examples of regulatory sequences include promoters,enhancers, transcription initiation sites, translation start sites,translation stop sites, and transcription terminators. The term“operably linked” refers to a juxtaposition of components such that theyare in a relationship permitting them to function in their intendedmanner. A regulatory sequence is “operably linked” to a coding regionwhen it is joined in such a way that expression of the coding region isachieved under conditions compatible with the regulatory sequence.

As used herein, an “exogenous polypeptide” and “exogenouspolynucleotide” refers to a polypeptide and polynucleotide,respectively, that is not normally or naturally found in a microbe,and/or has been introduced into a microbe. An exogenous polynucleotidemay be separate from the genomic DNA of a cell (e.g., it may be avector, such as a plasmid), or an exogenous polynucleotide may beintegrated into the genomic DNA of a cell. A regulatory region, such asa promoter, that is present in the genomic DNA of a microbe but has beenmodified to have a nucleotide sequence that is different from thepromoter normally present in the microbe is also considered an exogenouspolynucleotide. An exogenous polynucleotide may encode an exogenouspolypeptide or an endogenous polypeptide. For instance, a microbe may betransformed with a coding region that encodes a polypeptide that isnaturally expressed by the microbe. Such a polypeptide is endogenous tothat microbe, and it is encoded by an exogenous coding region. As usedherein, the term “endogenous polypeptide” and “endogenouspolynucleotide” refers to a polypeptide and polynucleotide,respectively, that is normally or naturally found in a microbe. An“endogenous polypeptide” is also referred to as a “native polypeptide,”and an “endogenous polynucleotide” is also referred to as a “nativepolynucleotide.”

The terms “complement” and “complementary” as used herein, refer to theability of two single stranded polynucleotides to base pair with eachother, where an adenine on one strand of a polynucleotide will base pairto a thymine or uracil on a strand of a second polynucleotide and acytosine on one strand of a polynucleotide will base pair to a guanineon a strand of a second polynucleotide. Two polynucleotides arecomplementary to each other when a nucleotide sequence in onepolynucleotide can base pair with a nucleotide sequence in a secondpolynucleotide. For instance, 5′-ATGC and 5′-GCAT are complementary. Theterm “substantial complement” and cognates thereof as used herein, referto a polynucleotide that is capable of selectively hybridizing to aspecified polynucleotide under stringent hybridization conditions.Stringent hybridization can take place under a number of pH, salt andtemperature conditions. The pH can vary from 6 to 9, preferably 6.8 to8.5. The salt concentration can vary from 0.15 M sodium to 0.9 M sodium,and other cations can be used as long as the ionic strength isequivalent to that specified for sodium. The temperature of thehybridization reaction can vary from 30° C. to 80° C., preferably from45° C. to 70° C. Additionally, other compounds can be added to ahybridization reaction to promote specific hybridization at lowertemperatures, such as at or approaching room temperature. Among thecompounds contemplated for lowering the temperature requirements isformamide. Thus, a polynucleotide is typically substantiallycomplementary to a second polynucleotide if hybridization occurs betweenthe polynucleotide and the second polynucleotide. As used herein,“specific hybridization” refers to hybridization between twopolynucleotides under stringent hybridization conditions.

As used herein, “genetically engineered microbe” and “microbe that hasbeen genetically engineered” refers to a microbe which has been altered“by the hand of man.” A genetically engineered microbe includes amicrobe into which has been introduced an exogenous polynucleotide,e.g., an expression vector. Genetically engineered microbe also refersto a microbe that has been genetically manipulated such that endogenousnucleotides have been altered to include a mutation, such as a deletion,an insertion, a transition, a transversion, or a combination thereof.For instance, an endogenous coding region could be deleted. Suchmutations may result in a polypeptide having a different amino acidsequence than was encoded by the endogenous polynucleotide. Anotherexample of a genetically engineered microbe is one having an alteredregulatory sequence, such as a promoter, to result in increased ordecreased expression of an operably linked endogenous coding region.

As used herein, “optimum growth temperature” and “T_(opt)” refer to theoptimal growth temperature of a microbe. The optimal growth temperatureof a microbe is the temperature at which the doubling time is theshortest. The T_(opt) of a thermophilic microbe is between 50° C. and nogreater than 75° C., and the T_(opt) of a hyperthermophilic microbe isbetween 75° C. and up to 100° C.

Conditions that are “suitable” for an event to occur, such as expressionof an exogenous polynucleotide in a cell to produce a polypeptide, orproduction of a product, or “suitable” conditions are conditions that donot prevent such events from occurring. Thus, these conditions permit,enhance, facilitate, and/or are conducive to the event.

The term “and/or” means one or all of the listed elements or acombination of any two or more of the listed elements.

The words “preferred” and “preferably” refer to embodiments of theinvention that may afford certain benefits, under certain circumstances.However, other embodiments may also be preferred, under the same orother circumstances. Furthermore, the recitation of one or morepreferred embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the invention.

The terms “comprises” and variations thereof do not have a limitingmeaning where these terms appear in the description and claims.

Unless otherwise specified, “a,” “an,” “the,” and “at least one” areused interchangeably and mean one or more than one.

Also herein, the recitations of numerical ranges by endpoints includeall numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.80, 4, 5, etc.).

For any method disclosed herein that includes discrete steps, the stepsmay be conducted in any feasible order. And, as appropriate, anycombination of two or more steps may be conducted simultaneously.

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The description that follows more particularly exemplifiesillustrative embodiments. In several places throughout the application,guidance is provided through lists of examples, which examples can beused in various combinations. In each instance, the recited list servesonly as a representative group and should not be interpreted as anexclusive list.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Enzymes and substrates reactions of the 3HP/4HB cycle in M.sedula. E1α, β, γ, acetyl/propionyl-CoA carboxylase; E2,malonyl/succinyl-CoA reductase; E3, malonate semialdehyde reductase; E4,3-hydroxypropionate:CoA ligase; E5, 3-hydroxypropionyl-CoA dehydratase;E6, acryloyl-CoA reductase; E7, methylmalonyl-CoA epimerase; E8,methylmalonyl-CoA mutase; E9, succinate semialdehyde reductase; E10,4-hydroxybutyrate-CoA ligase; E11, 4-hydroxybutyryl-CoA dehydratase;E12, crotonyl-CoA hydratase/(S)-3hydroxybutyryl-CoA dehydrogenase; E13,acetoacetyl-CoA β-ketothiolase.

FIG. 2. Bioreactor schematic for gas intensive fermentation of M.sedula. Tandem 2 L bioreactors, started at the same time with the sameseed inoculum, were used to grow M. sedula inside of a chemical fumehood. A solenoid valve on the H₂/CO₂ tank provided passive “fail-safe”operation by cutting off the flow of flammable gas in the event of foodfailure. Gas compositions for the three different conditions shownbottom right.

FIG. 3. 4-Hydroxybutryate-CoA synthetase candidates in M. sedula.Normalized transcription levels for M. sedula genes annotated as smallorganic acid or fatty-acid ligases and synthetases. High transcriptionlevels are shown in red, low transcription in green, correspondingnumbers represent least-squares means of normalized log 2-transformedtranscription levels relative to the overall average transcription levelof 0 (black). Conditions shown: (2010)—Heterotrophic, Autotrophic,Mixotrophic; (2012)—Autotrophic Carbon Limited (ACL), Autotrophic CarbonRich (ACR), Heterotrophic (HTR). Least-squares mean values are shownhere for ACL condition for these genes, along with the fold change ofgenes under ACL relative to HTR and their statistical significance. Allother microarray data can be found in the GEO deposit—GSE39944.

FIG. 4A-C. Specific activity of acyl-CoA ligases in the M. sedula carbonfixation pathway on various substrates. Specific activities of the newcandidates for 4-hydroxybutyrate-CoA ligase on a variety of substratescompared to reported data for Msed_1456, a 3-hydroxypropionate-CoAligase: Msed_0394 (A), Msed_0406 (B), and Msed_1456 (C). Msed_1456showed >1% activity on 3-hydroxybutyrate, but was not tested on4-hydroxybutyrate. Substrate abbreviations: ACE—acetate; PRO—propionate;3HP—3-hydroxypropionate; 4HB—4-hydroxybutyrate; BUT—butyrate;VAL—valerate.

FIGS. 5A-B. Specific activity of native Msed_1353 and Msed_1353-W424Gmutant on various substrates. Comparison of activity of Msed_1353 FIG.5A and Msed_1353-G424 FIG. 5B on a variety of short-chain linear organicacids. Substrate abbreviations: ACE—acetate; PRO—propionate;3HP—3-hydroxypropionate; 4HB—4-hydroxybutyrate; BUT—butyrate;VAL—valerate; HEX—hexanoate; OCT—octanoate.

FIG. 6. Reaction rate profile for acyl-CoA ligases. Michaelis-Mentenreaction rate curves shown with experimental data for Msed_0394(squares), Msed_0406 (circles), and Msed_1353-G424 (triangles) over arange of substrate concentrations.

FIGS. 7A-B. S. enterica acetyl-CoA synthetase (Acs) and Msed_0394 activesite comparison. Acs shown in gold (residue W414), Msed_0394 in cyan(residues W233, L307, V331, and P340). Ligand from Acs structure(adenosine-5′-propyl phosphate) is labeled Acs. FIG. 7A shows a sideview of binding pocket with inter-atomic distances given from phosphorusatom of propyl-phosphate moiety to select atom from amino acid residues.FIG. 7B shows an axial view from bottom of substrate binding pocket.

FIG. 8. Sequence alignment of S. enterica acetyl-CoA synthetase(STM4275) and M. sedula acyl-CoA ligases. Amino acid sequence alignmentof active site residues in putative acyl-CoA ligases reveals a conservedglycine (shown in red) except for Msed_1353, which has a tryptophanindicative of acetate-propionate CoA ligases. Alignment was generatedusing Chimera by superposition of I-TASSER 3D structural models.Consensus, SEQ ID NO:400; STM4275, SEQ ID NO:401 (IMB Gene ID number637214968); Msed_0394, SEQ ID NO:19 (IMB Gene ID number 640506300);Msed_0401, SEQ ID NO:20 (IMB Gene ID number 640506307); Msed_0406, SEQID NO:21 (IMB Gene ID number 640506312); Msed_1291, SEQ ID NO:22 (IMBGene ID number 640507181); Msed_1353, SEQ ID NO:23 (IMB Gene ID number640507242); and Msed_1422, SEQ ID NO:24 (IMB Gene ID number 640507311).

FIG. 9. SDS-PAGE gel images of purified recombinant enzymes. All sampleswere run on 4-12% NuPAGE® Bis-Tris Mini Gels (Life Technologies).BenchMark™ Protein Ladder (Invitrogen) was used for molecular weightreference.

FIG. 10. Progress curve for Msed_0406 on 4HB with controls. Examplereaction progress curve showing how data were generated for kineticcharacterization. Initial reaction rate was taken as the slope of thelinear region on the progress curve for a given substrate concentration(5 mM 4HB—circles). Two negative controls are shown: no 4HB (squares)and no ATP (triangles). A series of reaction rates was graphed oversubstrate concentration and a non-linear fitting was used to calculateMichaelis-Menten parameters.

FIGS. 11A-D. FIG. 11A shows the synthetic operon constructed to expressthe M. sedula genes encoding E1 (αβγ), E2 and E3 in P. furiosus underthe control of the promoter for the S-layer protein gene (P_(slp)). Thisincludes P. furiosus ribosomal binding sites (rbs) from highly-expressedgenes encoding pyruvate ferredoxin oxidoreductase subunit γ (porγ,PF0971), the S-layer protein (slp, PF1399) and cold-induced protein A(cipA, PF0190). FIG. 11B shows the first three enzymes of the M. sedula3-HP/4-HB cycle produce the key intermediate 3-hydroxypropionate (3-HP).E1 is acetyl/propionyl-CoA carboxylase (αβγ, encoded by Msed_0147,Msed_0148, Msed_1375): E2 is malonyl/succinyl-CoA reductase (Msed_0709)and E3 is malonate semialdehyde reductase (Msed_1993). NADPH isgenerated by P. furiosus soluble hydrogenase I (SH1), which reduces NADPwith hydrogen gas. FIG. 11C shows the first three enzymes (E1−E3) incontext of the complete 3-HP/4-HP cycle for carbon dioxide fixation byMetallosphaera sedula showing the three subpathways SP1, SP2, and SP3.At FIG. 11D the horizontal scheme shows the amount of energy (ATP),reductant (NADPH), oxidant (NAD) and CoASH required to generate one moleof acetyl-CoA from two moles of carbon dioxide.

FIGS. 12A-D. Temperature-dependent production of the SP1 pathway enzymesin P. furiosus strain PF506. FIG. 12A shows the growth of triplicatecultures at 98° C. (circles) and temperature (black line) for thetemperature shift from 98 to 75° C. are shown. FIG. 12B shows thesepcific activity (moles NADPH oxidized/min/mg) of the coupled activityof E2+E3 in cell-free extracts from cultures grown at 95° C. to a highcell density of 1×10⁸ cells/ml and then incubated for 18 hrs at theindicated temperature. FIG. 12C shows the activities of E1, E2+E3, andE1+E2+E3 after the temperature shift to 75° C. for the indicated timeperiod (see FIG. S4). The activities of a cell-free extract ofautotrophically-grown M. sedula cells is also shown (labeled Msed). Thespecific activities are: E1+E2+E3 coupled assay with acetyl-CoA andbicarbonate, E2+E3 coupled assay with malonyl-CoA, and E2 withsuccinyl-CoA as substrates. FIG. 12D shows the temperature dependence ofthe coupled activity of E2+E3 (circles) in the cell-free extracts afterinduction at 72° C. for 16 hr. The activity of P. furiosus glutamatedehydrogenase in the same cell-free extracts is also shown (squares).

FIGS. 13A-C. 3-HP production by P. furiosus. Cells were grown at 95° C.and then incubated at 72° C. for 16 hr to produce the SP1 enzymes. FIG.13A shows the in vitro 3-HP production from acetyl-CoA performed intriplicate. The sources of the C1 carbon (CO₂ or HCO₃ ⁻) and reducingequivalents (NADPH or NADP/H₂) are indicated. Rates are expressed asmoles of 3-HP produced/min/mg. FIG. 13B shows the in vivo 3-HPproduction by whole cells (static) using maltose as the source ofacetyl-CoA in the presence of hydrogen gas and bicarbonate using cellsgrown in a 100 ml sealed bottles without pH control. The P. furiosusstrains are MW56 (circles) and COM1 (squares). FIG. 13C shows the invivo 3-HP production by whole cells (stirred) of MW56 using maltose asthe source of acetyl-CoA (circles) and E2+E3 specific activity of thecell-free extracts (diamonds) using cells grown in a 20 L fermenter withpH control (6.8).

FIG. 14. Plasmid map of pALM506-1 used to transform P. furiosus strainΔpdaD to generate strain PF506.

FIG. 15. Plasmid map of pGL007 vector targeting the region betweenPF0574 and PF0575 in the P. furiosus genome.

FIG. 16. Plasmid map of pGL010 used to transform P. furiosus COM1 togenerate strain MW56.

FIG. 17. Growth of P. furiosus strain PF506 at 98° C. and subsequenttemperature shift to 75° C. P. furiosus was grown in four 800 mLcultures at 98° C. until the cell density reached 5×10⁸ cells/mL. Thetemperature (shown as black line) was then shifted to 75° C. andindividual bottles were removed and harvested after 0 (diamond), 16(square), 32 (triangle) and 48 (circle) hrs. The enzyme activities ineach cell type are summarized in FIG. 12B.

FIG. 18. Stability of E2 and E3 using an E2+E3 coupled assay at 75° C.after incubation at 90° C. for the indicated amount of time in cell-freeextracts of P. furiosus strain PF506 (circles) and of the endogenous P.furiosus glutamate dehydrogenase (squares). The specific activity ofE2+E3 in PF506 (grown at 72° C.) is about 2-fold higher than thatmeasured in M. sedula. Activity is expressed as percent of maximumactivity.

FIG. 19. Growth of P. furiosus COM 1, MW56 and PF506 during thetemperature shift from 98° C. to 70° C. Cell densities of COM1(diamonds), MW0056 (squares), and PF506 (triangles) are indicated. The400 mL cultures were grown at 95° C. for 9 hr and then allowed to coolat room temperature to 70° C. before being placed in a 70° C. incubator.

FIG. 20. Enzyme activities of E1 (left bar of each pair) and coupledE2+E3 (right bar of each pair) in cell-free extracts of the indicated P.furiosus strains after incubation at 70° C. for 16 hr, compared to thatmeasured for the cell-extract of autotrophically-grown M. sedula cells(labeled Msed).

FIGS. 21A-B. ESI-MS identification of 3-HP produced from acetyl-CoA, CO₂and H₂ (or NADPH) by cell-free extracts of P. furiosus strains ΔPdaDFIG. 21A) and PF506 FIG. 21B). The MS peak corresponding to the 3HPderivative (m/z 224, circled) was present above background only in therecombinant PF506 strain.

FIG. 22. Maltose and pyruvate metabolism by P. furiosus, and the keyroles of pyruvate ferredoxin oxidoreductase (POR) in acetyl-CoAproduction and of the membrane-bound hydrogenase (MBH) in H₂ production.

FIG. 23. In vivo production of 3-HP from maltose by whole cells of P.furiosus strain MW56 (left panel) and PF506 (right panel) after 10 min(blue) and 60 min (red) compared to a 1 mM 3-HP standard (black). Ablack arrow indicates the position of the 3-HP peaks. A total of 135 μMand 199 μM of 3-HP was produced by cell suspensions of MW56 (5×10¹⁰cells/mL) and of PF506 (5×10¹⁰ cells/mL), respectively, after 60 min at75° C.

FIG. 24. Design of an artificial operon encoding SP1 (E1−E3) forexpression in P. furiosus.

FIG. 25. SP1 expression cassette for cloning into pSPF300 vector. Thesequence of the SP1 expression cassette cloned into pSPF300 to makepALM506-1 is disclosed in Kelly et al. (WO 2013/067326).

FIG. 26. Construction of pALM506-1 plasmid for transformation of P.furiosus strain ΔpdaD (Kelly et al., WO 2013/067326)).

FIG. 27. Transcriptionally inactive zones for foreign gene insertion.

FIG. 28. Target genome regions in NCBI reference sequence versus COM1sequence.

FIG. 29. SOE-PCR products for constructing pGL002 and pGL007 targetinggenome regions 2 and 3. The nucleotide sequences of these are disclosedin Kelly et al. (WO 2013/067326)

FIG. 30. Construction of pGL002 vector targeting genome region 2.

FIG. 31. Construction of pGL007 vector targeting genome region 3.

FIG. 32. SP2B expression cassette for cloning into pGL002. The sequenceof P_(slp)-E7-E8α-E8β-E9γ expression cassette cloned into the Small siteof pGL002 is disclosed in Kelly et al. (WO 2013/067326).

FIG. 33. Construction of pGL005 vector for transformation of P. furiosusCOM1.

FIG. 34. SP1 expression cassette for cloning into pGL007. The sequenceof SP1 expression cassette cloned into pGL007 (genome region 3 insertionvector) to make pGL010) is disclosed in Kelly et al. (WO 2013/067326).

FIG. 35. Construction of pGL010 vector for transformation of COM1.

FIG. 36. NADPH-dependent assays for the E2, E2+E3 and E1+E2+E3 reactionsof SP1.

FIG. 37. NADPH-dependent assay for E9 of the SP2B subpathway.

FIG. 38. Growth of P. furiosus strain MW43 at 95° C. and temperatureshift from 65° C. to 90° C. for 18 hrs.

FIGS. 39A-C. E9 temperature profile and stability in cell-free extractsof P. furiosus strain MW43. FIG. 39A shows the E9 specific activity inMW43 versus Msed extract. FIG. 39B shows the E9 specific activity whenassayed at increasing temperatures. FIG. 39C shows the stability of E9over time when incubated at 90° C.

FIG. 40. Phosphate-dependent assay for E1.

FIG. 41. Scheme for producing acetyl CoA from pyruvate or maltose andfor producing ATP and NADPH for the SP1 pathway for 3-HP production bywhole cells of P. furiosus strains PF506 and MW56.

FIG. 42. Enzymes and substrates in final reactions of 3HP/4HB cycle inM. sedula. Enzymes: 10, 4-hydroxybutyrate-CoA synthetase; 11,4-hydroxybutyryl-CoA dehydratase; 12, crotonyl-CoAhydratase/(S)-3hydroxybutyryl-CoA dehydrogenase; 13, acetoacetyl-CoAβ-ketothiolase; 14 acetyl-CoA synthetase (non-native, used for HPLCassay).

FIGS. 43A-D. HPLC chromatograms demonstrating in vitro production ofacetate from 4-hydroxybutyrate. Samples and standards were derivatizedusing dibromoacetophenone (DBAP) and run on a reversed-phase column toshow production of acetate. Chromatograms shown are: FIG. 43A) 4HBstandard, FIG. 43B) 3HP standard, FIG. 43C) control reaction containingbuffer, cofactors, and 4HB but no enzymes, and FIG. 43D) SP3 reactionusing recombinant enzymes. Retention times: 6.9 min for 4HB and 9.1 minfor acetate.

FIG. 44. Transcriptional Heatmap for proposed 3HP/4HB cycle and centralmetabolism in M. sedula. Metabolic diagram shows 3HP/4HB pathway (topcenter), incomplete tricarboxylic acid cycle (TCA, center),gluconeogenesis (bottom center), and isoprenoid-based lipid biosynthesispathways (top left). Metabolic network adapted from Estelmann et. al.Enzymes: 1, acetyl-CoA/propionyl-CoA carboxylase; 2, malonyl-CoAreductase (NADPH); 3, malonic semialdehyde reductase (NADPH); 4, 3HP-CoAsynthetase (AMP-forming); 5, 3-hydroxypropionyl-CoA dehydratase; 6,acryloyl-CoA reductase (NADPH); 7, acetyl-CoA/propionyl-CoA carboxylase;8, methylmalonyl-CoA epimerase; 9, methylmalonyl-CoA mutase; 10,succinyl-CoA reductase (NADPH); 11, succinic semialdehyde reductase(NADPH); 12, 4HB-CoA synthetase (AMP-forming); 13, 4-hydroxybutyryl-CoAdehydratase; 14 and 15, crotonyl-CoA hydratase/(S)-3-hydroxybutyryl-CoAdehydrogenase (NAD⁺); 16, acetoacetyl-CoA b-ketothiolase; 17,succinyl-CoA synthetase (ADP-forming); 18, succinic semialdehydedehydrogenase; 19, succinate dehydrogenase; 20, fumarate hydratase; 21,malate dehydrogenase; 22, (si)-citrate synthase; 23, aconitase; 24,isocitrate dehydrogenase; 25, malic enzyme; 26, pyruvate: water dikinase(ATP); 27, PEP carboxylase; 28, PEP carboxykinase (GTP); 29, enolase;30, phosphoglycerate mutase; 31, phosphoglycerate kinase; 32,glyceraldehyde-3-phosphate dehydrogenase; 33, triosephosphate isomerase;34, fructose 1,6-bisphosphate aldolase/phosphatase; 35, malate synthase;36, acetyl-CoA acetyl-transferase; 37, HMG-CoA synthase; 38, HMG-CoAreductase. Abbreviations: Ac-CoA—acetyl-CoA, 3HP—3-hydroxypropionate;Suc-CoA—succinyl-CoA; Suc. semi.—succinic semialdehyde;4HB—4-hydroxybutyrate; AcAc-CoA—Acetoacetyl-CoA;HMG-CoA—3-hydroxy-3-methyl-glutaryl-CoA; PEP—phosphoenolpyruvate;F6P—Fructose-6-phosphate.

FIG. 45. Annotated 2-oxoacid oxidoreductases in M. sedula. Normalizedtranscription levels for M. sedula genes annotated as pyruvate (or2-oxoglutarate) flavodoxin/ferredoxin oxidoreductases. Hightranscription levels are shown in red, low transcription in green,corresponding numbers represent least-squares means of normalized log2-transformed transcription levels relative to the overall averagetranscription level of 0 (black). Annotations are from the Joint GenomeInstitute's Integrated Microbial Genome database (img.jgi.doe.gov).Conditions shown: Autotrophic Carbon Limited (ACL), Autotrophic CarbonRich (ACR), Heterotrophic (HTR). Fold change of gene transcription underACL relative to HTR and their statistical significance is also shown.All other microarray data can be found in the GEO deposit—GSE39944.

FIGS. 46A-D. Amino acid sequences of polypeptides that are part of the4-hydroxybutyrate cycle.

FIGS. 47A-F shows an amino acid alignment of Msed_0147 (SEQ ID NO:1) and18 other sequences. 640506050.Msed_0147 (SEQ ID NO:1),650848362.Ahos_2119 (SEQ ID NO:25), 650472093.SiRe_0254,643841501.YN1551_2862 (SEQ ID NO:26), 638192839.ST0593 (SEQ ID NO:402),2508723726.Met . . . 1DRAFT_00020780 (SEQ ID NO:28), 646527065.LD85 0260(SEQ ID NO:343), 2524413480.SacN8_01265 (SEQ ID NO:29),643828153.M1425_0254 (SEQ ID NO:30), 638197195.Saci_0260 (SEQ ID NO:31),643830840.LS215_0285 (SEQ ID NO:32), 643882885.M164_0272 (SEQ ID NO:33),650822319.Mcup_1926 (SEQ ID NO:34), 643836194.YG5714_0257 (SEQ IDNO:35), 638163678.SS02466 (SEQ ID NO:36), 650474851.SiH_0261 (SEQ IDNO:37), 646942932.Ssol_0270 (SEQ ID NO:38), 643842091.M1627_0254 (SEQ IDNO:39), 650023511.Sso198_010100000595 (SEQ ID NO:40).

FIGS. 48A-B. An amino acid alignment of Msed_0148 (SEQ ID NO:2) and 18other sequences. 640506051.Msed_0148 (SEQ ID NO:2), 638192838.ST0592(SEQ ID NO:41), 650822318.Mcup_1925 (SEQ ID NO:42), 2508723727.Met . . .1DRAFT_00020790 (SEQ ID NO:43), 650848361.Ahos_2118 (SEQ ID NO:344),638163677.SS_02464 (SEQ ID NO:44), 643842090.M1627_0253 (SEQ ID NO:45),646942931.Ssol_0269 (SEQ ID NO:46), 643828152.M1425_0253 (SEQ ID NO:47),643882884.M164_0271 (SEQ ID NO:112), 643830839.LS215_0284 (SEQ IDNO:48), 643841500.YN1551_2861 (SEQ ID NO:49), 643836193.YG5714_0256 (SEQID NO:50), 650472092.SiRe_0253 (SEQ ID NO:51), 650474850.SiH_0260 (SEQID NO:52), 650023512.Sso198_010100000600 (SEQ ID NO:53),646527064LD85_0259 (SEQ ID NO:54), 638197196.Saci_0261 (SEQ ID NO:55),2524413481.SacN8_01270 (SEQ ID NO:56).

FIGS. 49A-F. An amino acid alignment of Msed_01375 (SEQ ID NO:3) andother sequences. 640507264.Msed_1375 (SEQ ID NO:3), 638192837.ST0591(SEQ ID NO:57), 2508724172.Met . . . 1DRAFT_00025240 (SEQ ID NO:58),650821248.Mcup_0858 (SEQ ID NO:59), 646527063.LD85_0258 (SEQ ID NO:60),643842089.M1627_0252 (SEQ ID NO:61), 643841499.YN1551_2860 (SEQ IDNO:62), 643828151.M1425_0252 (SEQ ID NO:63), 643836192.YG5714_0255 (SEQID NO:64), 643830838LS215_0283 (SEQ ID NO:65), 646942930.Ssol_0268 (SEQID NO:66), 650023513.Sso198_010100000605 (SEQ ID NO:67),643882883.M164_0270 (SEQ ID NO:68), 650474849.SiH_0259 (SEQ ID NO:69),650472091.SiRe_0252 (SEQ ID NO:70), 650848360Ahos_2117 (SEQ ID NO:71),2524413482.SacN8 01275 (SEQ ID NO:72), 638197197.Saci_0262 (SEQ IDNO:73), 638163676.5502463 (SEQ ID NO:74).

FIGS. 50A-D. An amino acid alignment of Msed_0709 (SEQ ID NO:4) andother sequences. 640506613.Msed_0709 (SEQ ID NO:4), 638194641.ST2171(SEQ ID NO:75), 638199060.Saci_2147 (SEQ ID NO:76), 650848598.Ahos_2348(SEQ ID NO:77), 643833336.LS215_2961 (SEQ ID NO:78), 646945396.Ssol_2908(SEQ ID NO:79), 650025873.Sso198_010100012550 (SEQ ID NO:80),643885393.M164_2777 (SEQ ID NO:81), 643830551M1425_2796 (SEQ ID NO:82),643844535.M1627_2848 (SEQ ID NO:83), 2524415528.SacN8_11535 (SEQ IDNO:84), 650474573.SiRe_2691 (SEQ ID NO:85), 643841782.YN1551_3167 (SEQID NO:86), 2524415315.SacN8_10450 (SEQ ID NO:87), 638199277.Saci_2370(SEQ ID NO:88), 643838884.YG5714_2976 (SEQ ID NO:89), 650477400.SiH_2755(SEQ ID NO:90), 646529769.LD85_3126 (SEQ ID NO:91), 638163414.SS02178(SEQ ID NO:92), 2508722882.Met . . . 1DRAFT_00012340 (SEQ ID NO:93),650821817Mcup_1427 (SEQ ID NO:94).

FIGS. 51A-D. An amino acid alignment of Msed_1993 (SEQ ID NO:5) andother sequences. 640507881.Msed_1993 (SEQ ID NO:5), 2508724800.Met . . .1DRAFT_00031520 (SEQ ID NO:95), 650025277. Sso198_010100009521 (SEQ IDNO:96), 638161868.SS00647 (SEQ ID NO:97), 643829267.M1425_1490 (SEQ IDNO:98), 643837420.YG5714_1494 (SEQ ID NO:99), 643832024.LS215_1598 (SEQID NO:100), 643840152.YN1551_1342 (SEQ ID NO:101), 643884086.M164_1487(SEQ ID NO:102), 643843312.M1627_1605 (SEQ ID NO:103),650476065.SiH_1456 (SEQ ID NO:104), 646944319.Ssol_1706 (SEQ ID NO:105),650847331.Ahos_1103 (SEQ ID NO:106), 2524414811.SacN8_07880 (SEQ IDNO:107), 650473222.SiRe_1366 (SEQ ID NO:108), 646528384.LD85_1697 (SEQID NO:109), 638198535.Saci_1623 (SEQ ID NO:110), 638193893.ST1507 (SEQID NO:111), 650820669.Mcup_0293 (SEQ ID NO:113).

FIGS. 52A-N. An amino acid alignment of Msed_1456 (SEQ ID NO:6) andother sequences. 640507344.Msed_1456 (SEQ ID NO:6),650848309.Ahos_2066—(SEQ ID NO:114), 650821134.Mcup_0744 (SEQ IDNO:115), 638193050.ST0783 (SEQ ID NO:116), 639783349.Pisl 0270 (SEQ IDNO:117), 2508724181.Met . . . 1DRAFT_00025330 (SEQ ID NO:118),639773672.Tpen_0893 (SEQ ID NO:119), 650847233.Ahos_1005 (SEQ IDNO:120), 2505689392.Pyrfu_0975 (SEQ ID NO:121), 650025593.Sso198_010100011130 (SEQ ID NO:122), 638164412.SS03203 (SEQ ID NO:123),638198104.Saci_1184 (SEQ ID NO:124), 638171842.PAE2867 (SEQ ID NO:125),2524414384.SacN8_05775 (SEQ ID NO:126), 650473925.SiRe 2035 (SEQ IDNO:127), 643832752.LS21-5 2320 (SEQ ID NO:128), 650476750.S1H 21-03 (SEQID NO:129), 643884788.M164_2161 (SEQ ID NO:130), 643838221.YG5714 2284(SEQ ID NO:131), 646943552.Ssol 0940—(SEQ ID NO:132), 64383945 YN1-5510632 (SEQ ID NO:133), 643829949.M1425_2-157 (SEQ ID NO:134),643843952.M1627_2237 (SEQ ID NO:135), 646529117.LD85_2424 (SEQ IDNO:136).

FIGS. 53A-C. An amino acid alignment of Msed_2001 (SEQ ID NO:7) andother sequences. 640507889.Msed_2001 (SEQ ID NO:7), 638193901.ST1516(SEQ ID NO:138), 650847323.Ahos_1095 (SEQ ID NO:139),638198544.Saci_1633 (SEQ ID NO:140), 252441482.0.SacN807925 (SEQ IDNO:141), 650820662.Mcup_0286 (SEQ ID NO:142), 638161875.SS00654 (SEQ IDNO:143), 650025284.Sso198_010100009556 (SEQ ID NO:144), 2508724790.Met .. . 1DRAFT_00031420 (SEQ ID NO:145), 643843305.M1627_1597 (SEQ IDNO:146), 646528376.LD85_1689 (SEQ ID NO:147), 643840160.YN1551_1350 (SEQID NO:148), 643884079.M164_1479 (SEQ ID NO:149), 643832016.LS215_1590(SEQ ID NO:150), 643837412.YG5714_1486 (SEQ ID NO:151),646944326.Ssol_1713 (SEQ ID NO:152), 643829260.M1425_1482 (SEQ IDNO:153), 650476056.SiH_1448 (SEQ ID NO:154), 650473215.SiRe_1359 (SEQ IDNO:155).

FIGS. 54A-G. An amino acid alignment of Msed_1426 (SEQ ID NO:8) andother sequences. 640507315.Msed_1426 (SEQ ID NO:8), 638192718.ST0480(SEQ ID NO:156), 650821199.Mcup_0809 (SEQ ID NO:157),638197838.Saci_0911 (SEQ ID NO:158), 638161989.SS00764 (SEQ ID NO:159),643831892.LS215_1474 (SEQ ID NO:160), 643843128.M1627_1428 (SEQ IDNO:161), 643828184.M1425_0286 (SEQ ID NO:162), 650473079.SiRe_1239 (SEQID NO:163), 646528184.1 . . . D85_1501 (SEQ ID NO:164),643883965.M164_1370 (SEQ ID NO:165), 643842122.M1627_0286 (SEQ IDNO:166), 646942963.Ssol_0305 (SEQ ID NO:167), 643829150.M1425_1378 (SEQID NO:168), 650475916.SiH_1323 (SEQ ID NO:169), 643837294.YG5714_1372(SEQ ID NO:170), 646944442.Ssol 1823 (SEQ ID NO:171),643840284.YN1551_1469 (SEQ ID NO:172), 650848532.Ahos_2283 (SEQ IDNO:173), 2524414117.SacN8_04415 (SEQ ID NO:174),650025399.Sso198_010100010101 (SEQ ID NO:175), 2508722637.Met . . .1DRAFT00009890 (SEQ ID NO:176).

FIGS. 55A-B. An amino acid alignment of Msed_0639 (SEQ ID NO:9) andother sequences. 640506543.Msed_0639 (SEQ ID NO:9), 638192799.ST0554(SEQ ID NO:177), 638197850.Saci_0923 (SEQ ID NO:178),646527026.LD85_0221 (SEQ ID NO:179), 2524414129.SacN8_04475 (SEQ IDNO:180), 638163642.SS02426 (SEQ ID NO:181),650024093.Sso198_010100003518 (SEQ ID NO:182), 2508722799.Met . . .1DRAFT_00011510 (SEQ ID NO:183), 643882848.M164_0235 (SEQ ID NO:184),646942892.Ssol_0230 (SEQ ID NO:185), 643828115.M1425_0216 (SEQ IDNO:186), 650474810.SiH_0222 (SEQ ID NO:187), 643842053.M1627 0216 (SEQID NO:188), 650821907.Mcup_1517 (SEQ ID NO:189), 643830802.LS215_0247(SEQ ID NO:190), 643841463.YN1551_2823 (SEQ ID NO:191),650848464.Ahos_2217 (SEQ ID NO:192), 650472052.SiRe_0215 (SEQ IDNO:193), 643836157.YG57140220 (SEQ ID NO:194).

FIGS. 56A-L. An amino acid alignment of Msed_0638 (SEQ ID NO:10) andother sequences. 640506542.Msed_0638 (SEQ ID NO:10), 638192798.ST0552(SEQ ID NO:195), 638189489.APE1687 (SEQ ID NO:196), 650507277.VMUT_0924(SEQ ID NO:197), 2524414130.SacN8_04480 (SEQ ID NO:198),638197851.Saci_0924 (SEQ ID NO:199), 650821906.Mcup_1516 (SEQ IDNO:200), 648200341.Vdis_0037 (SEQ ID NO:201), 2508722798.Met . . .1DRAFT_00011500 (SEQ ID NO:202), 2510092565.Calag_0472 (SEQ ID NO:203),638163641.SS02425 (SEQ ID NO:204), 650024094.Sso198_010100003523 (SEQ IDNO:205), 646527025 LD85_0220 (SEQ ID NO:206), 643882847.M164_0234 (SEQID NO:207), 650472051.SiRe_0214 (SEQ ID NO:208), 643830801.LS215_0246(SEQ ID NO:209), 646942891.Ssol 0229 (SEQ ID NO:210),650848463.Ahos_2216 (SEQ ID NO:211), 643836156.YG5714_0219 (SEQ IDNO:212), 643828114.M1425_0215 (SEQ ID NO:213), 643841462.YN1551_2822(SEQ ID NO:214), 650474809.SiH_0221 (SEQ ID NO:215),643842052.M1627_0215 (SEQ ID NO:216), 648118006.ASAC 1077 (SEQ IDNO:217).

FIGS. 57A-B. An amino acid alignment of Msed_2055 (SEQ ID NO:11) andother sequences. 640507945.Msed_2055 (SEQ ID NO:11), 638194564ST2096(SEQ ID NO:218), 638189488 APE1686 (SEQ ID NO:219), 650471903 SiRe_0075(SEQ ID NO:220), 650474657 SiH_0076 (SEQ ID NO:221),643827976.M1425_0076 (SEQ ID NO:222), 646942747 Ssol_0081 (SEQ IDNO:223), 643882689.M164_0076 (SEQ ID NO:224), 643841912M1627_0076 (SEQID NO:226), 6438306311S215_0076 (SEQ ID NO:227), 646526885LD85_0076 (SEQID NO:228), 643838963.YN1551_0076 (SEQ ID NO:229), 643836014.YG5714_0078(SEQ ID NO:230), 6482.00342.Vdis_0038 (SEQ ID NO:231),650820610.Mcup_0235 (SEQ ID NO:232), 2510092566.Calag.0473 (SEQ IDNO:233), 250872472814et . . . IDRAFT_00030800 (SEQ ID NO:234),638197003.Saci_0062 (SEQ ID NO:235), 2524413284.SacN8_00295 (SEQ IDNO:236), 650025504 Sso198_010100010675 (SEQ ID NO:237), 650846706Abos_0509 (SEQ ID NO:238), 638163495.SS02266 (SEQ ID NO:239),648118007ASAC_1078 (SEQ ID NO:240), 650507278.VMUT_0925 (SEQ ID NO:241).

FIGS. 58A-D. An amino acid alignment of Msed_1424 (SEQ ID NO:12) andother sequences. 640507313.Msed_1424 (SEQ ID NO:12), 638194516.ST2056(SEQ ID NO:242), 2524415313.SacN8_10440 (SEQ ID NO:243),638161699.SS00472 (SEQ ID NO:244), 646944079.Ssol_1454 (SEQ ID NO:245),643829410.M1425_1632 (SEQ ID NO:246), 643839999.YN1551_1180 (SEQ IDNO:247), 643884283.M164_1679 (SEQ ID NO:248), 643837646.YG5714_1723 (SEQID NO:249), 643832179.LS215_1759 (SEQ ID NO:250), 643843451.M1627_1747(SEQ ID NO:251), 650473386.SiRe_1527 (SEQ ID NO:252),650848525.Ahos_2277 (SEQ ID NO:253), 650476220.SiH_1606 (SEQ ID NO:254),646528572.LD85_1888 (SEQ ID NO:255), 638199058.Saci_2145 (SEQ IDNO:256), 650821201.Mcup_0811 (SEQ ID NO:257),650024197.Sso198_010100004040 (SEQ ID NO:258), 2508722628.Met . . .1DRAFT_00009800 (SEQ ID NO:259).

FIGS. 59A-O. An amino acid alignment of Msed_0406 (SEQ ID NO:14) andother sequences. 640506312.Msed_0406 (SEQ ID NO:14), 638193516.ST1190(SEQ ID NO:345), 638195071.ST2575 (SEQ ID NO:346), 641669006.Tneu_1843(SEQ ID NO:347), 650847233.Ahos_1005 (SEQ ID NO:348),650821270.Mcup_0880 (SEQ ID NO:260), 638163277.SS02041 (SEQ ID NO:261),638163135.SS01903n (SEQ ID NO:262), 638198069.Saci_1149 (SEQ ID NO:263),650772447.TUZN 2145 (SEQ ID NO:264), 648117514.ASAC_0597 (SEQ IDNO:265), 2508723436.Met . . . IDRAFT_00017880 (SEQ ID NO:266),643840674.YN1551_1878 (SEQ ID NO:267), 643883328.M164 0732 (SEQ IDNO:268), 650472518.SiRe_0686 (SEQ ID NO:269), 2524414348.SacN8_05595(SEQ ID NO:270), 646527485.LD85_0753 (SEQ ID NO:271),646945208.Ssol_2702 (SEQ ID NO:272), 650822064.Mcup_1674 (SEQ IDNO:273), 643828660.M1425_0851 (SEQ ID NO:274), 643831217.LS215 0753 (SEQID NO:275), 650024986.Sso198_010100008026 (SEQ ID NO:276),643831373.LS215_0923 (SEQ ID NO:277), 643828535.M1425_0704 (SEQ IDNO:278), 650475223.SiH_0646 (SEQ ID NO:279), 643842477.M1627_0708 (SEQID NO:349), 643836911.YG5714_0994 (SEQ ID NO:350), 650508650.VMUT 2290(SEQ ID NO:351).

FIGS. 60A-K. An amino acid alignment of Msed_1321 (SEQ ID NO:15) andother sequences. 640507210.Msed_1321 (SEQ ID NO:15), 650821278.Mcup_0888 (SEQ ID NO:352), 2511627386.1TX_1102 (SEQ ID NO:353),650848279.Ahos_2036 (SEQ ID NO:355), 650024685.Sso198_010100006527 (SEQID NO:356), 638163940.SS02738 (SEQ ID NO:357), 638171713.PAE2693 (SEQ IDNO:358), 638194068ST1659 (SEQ ID NO:359), 2508721913.Met . . .1DRAFT_00002650 (SEQ ID NO:360), 2512378777.Pogu_1298 (SEQ ID NO:361),638199056.Saci_2143 (SEQ ID NO:362), 2524415311.SacN810430 (SEQ IDNO:363), 640125146.Pca1_1396 (SEQ ID NO:364), 641667580.Tneu_0422 (SEQID NO:365), 2511694157.P186_0718 (SEQ ID NO:366), 643833127.LS215_2744(SEQ ID NO:367), 643830347341425_2585 (SEQ ID NO:368),643844328M1627_2638 (SEQ ID NO:369), 639783328.Pisl_0248 (SEQ IDNO:370), 650477167.S1H_2522 (SEQ ID NO:371), 643839021.YN1551_0139 (SEQID NO:372), 640897163.Igni_0595 (SEQ ID NO:373), 643885186.M164_2569(SEQ ID NO:374), 650474246.SiRe_2362 (SEQ ID NO:375),643838664.YG5714_2751 (SEQ ID NO:376), 646943192.Ssol_0550 (SEQ IDNO:377), 646529546.LD85 2896 (SEQ ID NO:378).

FIGS. 61A-G. An amino acid alignment of Msed_0399 (SEQ ID NO:16) andother sequences. 640506305.Msed_0399 (SEQ ID NO:16), 2508723442.Met . .. 1DRAFT_00017940 (SEQ ID NO:379), 648117957.ASAC_1031 (SEQ ID NO:380),638170750.PAE1383 (SEQ ID NO:381), 643841549.YN1551_2911 (SEQ IDNO:382), 643882933.M164_0319 (SEQ ID NO:383), 643842139.M1627_0303 (SEQID NO:384), 641667694.Tneu_0541 (SEQ ID NO:385), 643828200.M1425_0302(SEQ ID NO:386), 650472146.SiRe_0307 (SEQ ID NO:387), 650474900.SiH_0309(SEQ ID NO:388), 646942979.Ssol_0321 (SEQ ID NO:389), 639784518Pisl_1434(SEQ ID NO:390), 640468018.Pars_0453 (SEQ ID NO:391),640897631.Igni_1058 (SEQ ID NO:392), 646527111 LD85_0308 (SEQ IDNO:393), 650023499.Sso198_010100000535 (SEQ ID NO:394),638163722.SS02514 (SEQ ID NO:395), 638196031.Saci_1109 (SEQ ID NO:396),2524414310.SacN8_05395 (SEQ ID NO:397), 638189274 APE1484 (SEQ IDNO:398), 65082207014cup_1680 (SEQ ID NO:399).

FIGS. 62A-I. An amino acid alignment of Msed_0656 (SEQ ID NO:17) andother sequences. 640506560Msed_0656 (SEQ ID NO:17), 638192756.ST0514(SEQ ID NO:280), 650770713.TUZN_0403 (SEQ ID NO:281),650821827.Mcup_1437 (SEQ ID NO:282), 646942850Ssol_0188 (SEQ ID NO:283),643841420.YN1551_2779 (SEQ ID NO:284), 650848421.Ahos_2176 (SEQ IDNO:285), 641667412.Tneu_0249 (SEQ ID NO:286), 638197890.Saci_0963 (SEQID NO:287), 650472007.SiRe_0173 (SEQ ID NO:288), 643836114.YG5714_0177(SEQ ID NO:289), 643842011.M1627_0173 (SEQ ID NO:290),650474766.SiH_0179 (SEQ ID NO:291), 643830761_LS215_0204 (SEQ IDNO:292), 640124530.Pca1_0781 (SEQ ID NO:293), 640467886.Pars_0309 (SEQID NO:294), 643882805.M164_0192 (SEQ ID NO:295), 640897989.Igni_1401(SEQ ID NO:296), 2512379618.Pogu_2093 (SEQ ID NO:297), 646526983.LD850177 (SEQ ID NO:298), 643828073M1425_0173 (SEQ ID NO:299),2524414169.SacN8_04675 (SEQ ID NO:300), 2508722819.Met . . . 1DRAFT00011710 (SEQ ID NO:301), 2511627177.TTX_0886 (SEQ ID NO:302),650024136.Sso198_010100003733 (SEQ ID NO:303), 638163597.SS02377 (SEQ IDNO:304), 648117228.ASAC_0321 (SEQ ID NO:305).

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Provided herein are systems for sequestering carbon dioxide from theatmosphere using hydrogen gas as the driving force to convert the carbonto C2, C3, and C4 compounds, including compounds useful in theproduction of biofuels and plastics. In one embodiment, the system is acomplete cycle. This cycle, also referred to herein as the4-hydroxybutyrate cycle, can be broken down into three sub-pathways, asshown in equations 1-3,

Acetyl CoA+CO₂+ATP+2H₂→3-HP+ADP+Pi+CoA  [1]

3-HP+CO₂+2ATP+3H2→4-HB+ADP+AMP+Pi+PPi  [2]

4-HB+ATP+NAD++2CoA→2Acetyl CoA+AMP+PPi+NADH  [3]

where 3-HP is 3-hydroxypropionate, and 4-HB is 4-hydroxybutyrate. Thereaction described in equation 1 is also referred to herein as the 3-HPsubpathway or SP1, and the reaction described in equation 2 is alsoreferred to herein as the 4-HB subpathway or SP2. Thus, the systemdescribed herein can be used to produce 3-HP, 4-HB, acetyl CoA, or acombination thereof. In some embodiments other compounds may beproduced, as described in greater detail herein.

In one embodiment, which is described by equation 1 and shown in FIG. 1as catalyzed by enzymes E1α, β, γ, E2, and E3, the system includes apolypeptide having acetyl/propionyl-CoA carboxylase activity (E1α, β,γ), a polypeptide having malonyl/succinyl-CoA reductase activity (E2),and a polypeptide having malonate semialdehyde reductase activity (E3).In one aspect of this embodiment, the system produces 3-HP. Aspects ofthe production of 3-HP, including useful carbon donors and electrondonors, are discussed herein.

A polypeptide having acetyl/propionyl-CoA carboxylase activity means thepolypeptide catalyzes the conversion of acetyl CoA to malonyl-CoA or theconversion of propionyl-CoA to (S)-methylmalonyl-CoA. Theacetyl/propionyl-CoA carboxylase activity of a polypeptide may bedetermined by routine methods known in the art.

An example of a polypeptide having acetyl/propionyl-CoA carboxylaseactivity is a heterotrimeric polypeptide that includes one amino acidsequence encoded by coding sequence Msed_0147 of Genbank accessionNC_009440 and disclosed at SEQ ID NO:1, one amino acid sequence encodedby coding sequence Msed_0148 of Genbank accession NC_009440 anddisclosed at SEQ ID NO:2, and one amino acid sequence encoded by codingsequence Msed_1375 of Genbank accession NC_009440 and disclosed at SEQID NO:3.

Other examples of polypeptides having acetyl/propionyl-CoA carboxylaseactivity include a polypeptide having structural similarity to the aminoacid sequence encoded by coding sequence Msed_0147 of Genbank accessionNC_009440 and disclosed at SEQ ID NO:1, a polypeptide having structuralsimilarity to the amino acid sequence encoded by coding sequenceMsed_0148 of Genbank accession NC_009440 and disclosed at SEQ ID NO:2,and/or a polypeptide having structural similarity to the amino acidsequence encoded by coding sequence Msed_1375 of Genbank accessionNC_009440 and disclosed at SEQ ID NO:3. A candidate polypeptide havingstructural similarity to one of the polypeptides SEQ ID NO:1, 2, or 3has acetyl/propionyl-CoA carboxylase activity when expressed in amicrobe with the other 2 reference polypeptides. For instance, whendetermining if a candidate polypeptide having some level of identity toSEQ ID NO:1 has acetyl/propionyl-CoA carboxylase activity, the candidatepolypeptide is expressed in a microbe with reference polypeptides SEQ IDNO:2 and 3. When determining if a candidate polypeptide having somelevel of identity to SEQ ID NO:2 has acetyl/propionyl-CoA carboxylaseactivity, the candidate polypeptide is expressed in a microbe withreference polypeptides SEQ ID NO:1 and 3. When determining if acandidate polypeptide having some level of identity to SEQ ID NO:3 hasacetyl/propionyl-CoA carboxylase activity, the candidate polypeptide isexpressed in a microbe with reference polypeptides SEQ ID NO:1 and 2.

Additional examples of polypeptides expected to haveacetyl/propionyl-CoA carboxylase activity may be obtained from membersof the orders Sulfolobaceae (such as Metallosphaera sedula DSM 5348, M.yellowstonensis, M. prunae, and M. cuprina Ar-4, Acidianus hospitalisW1, Sulfolobus tokodaii str. 7, S. acidocaldarius DSM 639, S. islandicusY.G.57.14, S. islandicus Y.N.15.51, S. islandicus L.S.2.15, S.islandicus L.D.8.5, S. islandicus M.16.4, S. solfataricus P2, and S.islandicus M.14.25) and Chloroflexales (such as Chloroflexus sp.Y-400-fl, C. aurantiacus J-10-fl, and C. aggregans DSM 9485).

A polypeptide having malonyl/succinyl-CoA reductase activity means thepolypeptide catalyzes the conversion of malonyl-CoA to malonatesemialdehyde or succinyl-CoA to succinate semialdehyde. Themalonyl/succinyl-CoA reductase activity of a polypeptide may bedetermined by routine methods known in the art. An example of such apolypeptide includes an amino acid sequence encoded by coding sequenceMsed_0709 of Genbank accession NC_009440 and disclosed at SEQ ID NO:4.

Other examples of polypeptides having malonyl/succinyl-CoA reductaseactivity include a polypeptide having structural similarity to the aminoacid sequence encoded by coding sequence Msed_0709 of Genbank accessionNC_009440 and disclosed at SEQ ID NO:4.

Additional examples of polypeptides expected to havemalonyl/succinyl-CoA reductase activity may be obtained from members ofthe orders Sulfolobaceae (such as Metallosphaera sedula DSM 5348, M.yellowstonensis, M. prunae, and M. cuprina Ar-4, Acidianus hospitalisW1, Sulfolobus tokodaii str. 7, S. acidocaldarius DSM 639, S. islandicusY.G.57.14, S. islandicus Y.N.15.51, S. islandicus L.S.2.15, S.islandicus L.D.8.5, S. islandicus M.16.4, S. solfataricus P2, and S.islandicus M.14.25) and Desulfurococcales (such as Ignicoccus hospitalisKIN4/I) and Euryarchaeotes (Thermococcales) (such as Pyrococcus sp.NA2), and Chloroflexales (such as Chloroflexus sp. Y-400-fl, C.aurantiacus J-10-fl, and C. aggregans DSM 9485).

A polypeptide having malonate semialdehyde reductase activity means thepolypeptide catalyzes the conversion of malonate semialdehyde to3-hydroxypropionate. The malonate semialdehyde reductase activity of apolypeptide may be determined by routine methods known in the art. Anexample of such a polypeptide includes one amino acid sequence encodedby coding sequence Msed_1993 of Genbank accession NC_009440 anddisclosed at SEQ ID NO:5.

Other examples of polypeptides having malonate semialdehyde reductaseactivity include a polypeptide having structural similarity to the aminoacid sequence encoded by coding sequence Msed_1993 of Genbank accessionNC_009440 and disclosed at SEQ ID NO:5.

Additional examples of polypeptides expected to have malonatesemialdehyde reductase activity may be obtained from members of theorder Sulfolobaceae (such as Metallosphaera sedula DSM 5348, M.yellowstonensis, M. prunae, and M. cuprina Ar-4, Acidianus hospitalisW1, Sulfolobus tokodaii str. 7, S. acidocaldarius DSM 639, S. islandicusY.G.57.14, S. islandicus Y.N.15.51, S. islandicus L.S.2.15, S.islandicus L.D.8.5, S. islandicus M.16.4, S. solfataricus P2, and S.islandicus M.14.25).

In one embodiment, which is described by equation 2 and shown in FIG. 1as catalyzed by enzymes E4, E5, E6, E7, and E8α and β, the systemincludes a polypeptide having 3-hydroxypropionate:CoA ligase activity(E4), a polypeptide having 3-hydroxypropionyl-CoA dehydratase activity(E5), a polypeptide having acryloyl-CoA reductase activity (E6), apolypeptide having methylmalonyl-CoA epimerase activity (E7), apolypeptide having methylmalonyl-CoA mutase activity (E8αβ), and apolypeptide having succinate semialdehyde reductase activity (E9). Inone aspect of this embodiment, the system produces 4-HB. The system mayalso include a polypeptide having acetyl/propionyl-CoA carboxylaseactivity (E1α, β, γ), a polypeptide having malonyl/succinyl-CoAreductase activity (E2), polypeptides which are described above. Aspectsof the production of 4-HB, including useful carbon donors and electrondonors, are discussed herein.

A polypeptide having 3-hydroxypropionate:CoA ligase activity means thepolypeptide catalyzes the conversion of 3-hydroxypropionate to3-hydroxypropionyl CoA. The 3-hydroxypropionate:CoA ligase activity of apolypeptide may be determined by routine methods known in the art. Anexample of such a polypeptide includes an amino acid sequence encoded bycoding sequence Msed_1456 of Genbank accession NC_009440 and disclosedat SEQ ID NO:6.

Other examples of polypeptides having 3-hydroxypropionate:CoA ligaseactivity include a polypeptide having structural similarity to the aminoacid sequence encoded by coding sequence Msed_1456 of Genbank accessionNC_009440 and disclosed at SEQ ID NO:6.

Additional examples of polypeptides expected to have3-hydroxypropionate:CoA ligase activity may be obtained from members ofthe orders Sulfolobaceae (such as Metallosphaera sedula DSM 5348, M.yellowstonensis, M. prunae, and M. cuprina Ar-4, Acidianus hospitalisW1, Sulfolobus tokodaii str. 7, S. acidocaldarius DSM 639, S. islandicusY.G.57.14, S. islandicus Y.N.15.51, S. islandicus L.S.2.15, S.islandicus L.D.8.5, S. islandicus M.16.4, S. solfataricus P2, and S.islandicus M.14.25), Thermoproteales (such as Vulcanisaeta moutnovskia768-28 and V. distributa DSM 14429), Acidilobales (such as Acidilobussaccharovorans 345-15), and Euryarchaeotes (Thermococcales) (such asThermococcus sibiricus MM 739, T. barophilus MP, Pyrococcus furiosus DSM3638, Pyrococcus sp. NA2, P. horikoshii OT3, Thermococcus gammatoleransEJ3).

A polypeptide having 3-hydroxypropionyl-CoA dehydratase activity meansthe polypeptide catalyzes the conversion of 3-hydroxypropionyl-CoA toacryloyl-CoA. The 3-hydroxypropionyl-CoA dehydratase activity of apolypeptide may be determined by routine methods known in the art. Anexample of such a polypeptide includes an amino acid sequence encoded bycoding sequence Msed_2001 of Genbank accession NC_009440 and disclosedat SEQ ID NO:7.

Other examples of polypeptides having 3-hydroxypropionyl-CoA dehydrataseactivity include a polypeptide having structural similarity to the aminoacid sequence encoded by coding sequence Msed_2001 of Genbank accessionNC_009440 and disclosed at SEQ ID NO:7.

Additional examples of polypeptides expected to have3-hydroxypropionyl-CoA dehydratase activity may be obtained from membersof the orders Sulfolobaceae (such as Metallosphaera sedula DSM 5348, M.yellowstonensis, M. prunae, and M. cuprina Ar-4, Acidianus hospitalisW1, Sulfolobus tokodaii str. 7, S. acidocaldarius DSM 639, S. islandicusY.G.57.14, S. islandicus Y.N.15.51, S. islandicus L.S.2.15, S.islandicus L.D.8.5, S. islandicus M.16.4, S. solfataricus P2, and S.islandicus M.14.25), Thermoproteales (such as Vulcanisaeta distributaDSM 14429), Acidilobales (such as Acidilobus saccharovorans 345-15), andDesulfurococcales (such as Aeropyrum pernix K1).

A polypeptide having acryloyl-CoA reductase activity means thepolypeptide catalyzes the conversion of acryloyl-CoA to propionyl-CoA.The acryloyl-CoA reductase activity of a polypeptide may be determinedby routine methods known in the art. An example of such a polypeptideincludes an amino acid sequence encoded by coding sequence Msed_1426 ofGenbank accession NC_009440 and disclosed at SEQ ID NO:8.

Other examples of polypeptides having acryloyl-CoA reductase activityinclude a polypeptide having structural similarity to the amino acidsequence encoded by coding sequence Msed_1426 of Genbank accessionNC_009440 and disclosed at SEQ ID NO:8.

Additional examples of polypeptides expected to have acryloyl-CoAreductase activity may be obtained from members of the ordersSulfolobaceae (such as Metallosphaera sedula DSM 5348, M.yellowstonensis, M. prunae, and M. cuprina Ar-4, Acidianus hospitalisW1, Sulfolobus tokodaii str. 7, S. acidocaldarius DSM 639, S. islandicusY.G.57.14, S. islandicus Y.N.15.51, S. islandicus L.S.2.15, S.islandicus L.D.8.5, S. islandicus M.16.4, S. solfataricus P2, and S.islandicus M.14.25), and Thermoproteales (such as Vulcanisaetamoutnovskia 768-28 and V. distributa DSM 14429).

A polypeptide having methylmalonyl-CoA epimerase activity means thepolypeptide catalyzes the conversion of (S)-methylmalonyl-CoA to(R)-methylmalonyl-CoA. The methylmalonyl-CoA epimerase activity of apolypeptide may be determined by routine methods known in the art. Anexample of such a polypeptide includes an amino acid sequence encoded bycoding sequence Msed_0639 of Genbank accession NC_009440 and disclosedat SEQ ID NO:9.

Other examples of polypeptides having methylmalonyl-CoA epimeraseactivity include a polypeptide having structural similarity to the aminoacid sequence encoded by coding sequence Msed_0639 of Genbank accessionNC_009440 and disclosed at SEQ ID NO:9.

Additional examples of polypeptides expected to have methylmalonyl-CoAepimerase activity may be obtained from members of the ordersSulfolobaceae (such as Metallosphaera sedula DSM 5348, M.yellowstonensis, M. prunae, and M. cuprina Ar-4, Acidianus hospitalisW1, Sulfolobus tokodaii str. 7, S. acidocaldarius DSM 639, S. islandicusY.G.57.14, S. islandicus Y.N.15.51, S. islandicus L.S.2.15, S.islandicus L.D.8.5, S. islandicus M.16.4, S. solfataricus P2, and S.islandicus M.14.25), Thermoproteales (such as Vulcanisaeta distributaDSM 14429), Euryarchaeotes (Thermococcales) (such as Thermococcussibiricus MM 739, T. barophilus MP, Pyrococcus furiosus DSM 3638,Pyrococcus sp. NA2, P. horikoshii OT3, T. gammatolerans EJ3, P. abyssiGE5, and Thermococcus onnurineus NA1), and Chloroflexales (such asChloroflexus sp. Y-400-fl, C. aurantiacus J-10-fl, and C. aggregans DSM9485).

An example of a polypeptide having methylmalonyl-CoA mutase activity isa heterodimeric polypeptide that includes one amino acid sequenceencoded by coding sequence Msed_0638 of Genbank accession NC_009440 anddisclosed at SEQ ID NO:10, and one amino acid sequence encoded by codingsequence Msed_2055 of Genbank accession NC_009440 and disclosed at SEQID NO:11.

Other examples of polypeptides having methylmalonyl-CoA mutase activityinclude a polypeptide having structural similarity to the amino acidsequence encoded by coding sequence Msed_0638 of Genbank accessionNC_009440 and disclosed at SEQ ID NO:10, and/or a polypeptide havingstructural similarity to the amino acid sequence encoded by codingsequence Msed_2055 of Genbank accession NC_009440 and disclosed at SEQID NO:11. A candidate polypeptide having structural similarity to one ofthe polypeptides SEQ ID NO:10 or 11 has methylmalonyl-CoA mutaseactivity when expressed in a microbe with the other referencepolypeptide. For instance, when determining if a candidate polypeptidehaving some level of identity to SEQ ID NO:10 has methylmalonyl-CoAmutase activity, the candidate polypeptide is expressed in a microbewith reference polypeptide SEQ ID NO:11. When determining if a candidatepolypeptide having some level of identity to SEQ ID NO:11 hasmethylmalonyl-CoA mutase activity, the candidate polypeptide isexpressed in a microbe with reference polypeptide SEQ ID NO:10.

Additional examples of polypeptides expected to have methylmalonyl-CoAmutase activity may be obtained from members of the orders Sulfolobaceae(such as Metallosphaera sedula DSM 5348, M. yellowstonensis, M. prunae,and M. cuprina Ar-4, Acidianus hospitalis W1, Sulfolobus tokodaii str.7, S. acidocaldarius DSM 639, S. islandicus Y.G.57.14, S. islandicusY.N.15.51, S. islandicus L.S.2.15, S. islandicus L.D.8.5, S. islandicusM.16.4, S. solfataricus P2, and S. islandicus M.14.25), Thermoproteales(such as Vulcanisaeta moutnovskia 768-28 and V. distributa DSM 14429),Acidilobales (such as Acidilobus saccharovorans 345-15),Desulfurococcales (such as Aeropyrum pernix K1), Euryarchaeotes(Thermococcales) (such as Thermococcus sibiricus MM 739, T. barophilusMP, Pyrococcus furiosus DSM 3638, Pyrococcus sp. NA2, P. horikoshii OT3,T. gammatolerans EJ3, P. abyssi GE5, and Thermococcus onnurineus NA1),and Chloroflexales (such as Chloroflexus sp. Y-400-fl, C. aurantiacusJ-10-fl, and C. aggregans DSM 9485).

A polypeptide having succinate semialdehyde reductase activity means thepolypeptide catalyzes the conversion of succinate semialdehyde to4-hydroxybutyrate. The succinate semialdehyde reductase activity of apolypeptide may be determined by routine methods known in the art. Anexample of such a polypeptide includes an amino acid sequence encoded bycoding sequence Msed_1424 of Genbank accession NC_009440 and disclosedat SEQ ID NO:12.

Other examples of polypeptides having succinate semialdehyde reductaseactivity include a polypeptide having structural similarity to the aminoacid sequence encoded by coding sequence Msed_1424 of Genbank accessionNC_009440 and disclosed at SEQ ID NO:12.

Additional examples of polypeptides expected to have semialdehydereductase activity may be obtained from members of the orderSulfolobaceae (such as Metallosphaera sedula DSM 5348, M.yellowstonensis, M. prunae, and M. cuprina Ar-4, Acidianus hospitalisW1, Sulfolobus tokodaii str. 7, S. acidocaldarius DSM 639, S. islandicusY.G.57.14, S. islandicus Y.N.15.51, S. islandicus L.S.2.15, S.islandicus L.D.8.5, S. islandicus M.16.4, S. solfataricus P2, and S.islandicus M.14.25).

In one embodiment, which is described by equation 3 and shown in FIG. 1as catalyzed by enzymes E10, E11, E12, and E13, the system includes apolypeptide having a polypeptide having 4-hydroxybutyrate:CoA ligaseactivity (E10), a polypeptide having 4-hydroxybutyrl-CoA dehydrataseactivity (E11), a polypeptide having crotonyl-CoAhydratase/(S)-3-hydroxybutyrl-CoA dehydrogenase activity (E12), and apolypeptide having acetoacetyl-CoA β-ketothiolase activity (E13). In oneaspect of this embodiment, the system produces acetyl-CoA. Aspects ofthe production of acetyl-CoA, including useful carbon donors andelectron donors, are discussed herein.

A polypeptide having 4-hydroxybutyrate:CoA ligase activity means thepolypeptide catalyzes the conversion of 4-hydroxybutyrate to4-hydroxybutyryl-CoA. The 4-hydroxybutyrate:CoA ligase activity of apolypeptide may be determined by routine methods known in the art. Anexample of such a polypeptide includes an amino acid sequence encoded bycoding sequence Msed_0394 of Genbank accession NC_009440 and disclosedat SEQ ID NO:13. Another example of a polypeptide having4-hydroxybutyrate:CoA ligase activity includes an amino acid sequenceencoded by coding sequence Msed_0406 of Genbank accession NC_009440 anddisclosed at SEQ ID NO:14.

Other examples of polypeptides having 4-hydroxybutyrate:CoA ligaseactivity include a polypeptide having structural similarity to the aminoacid sequence encoded by coding sequence Msed_0394 of Genbank accessionNC_009440 and disclosed at SEQ ID NO:13 and a polypeptide havingstructural similarity to the amino acid sequence encoded by codingsequence Msed_0406 of Genbank accession NC_009440 and disclosed at SEQID NO:14.

In one embodiment, an example of a polypeptide having4-hydroxybutyrate:CoA ligase activity is an amino acid sequence encodedby coding sequence Msed_1353 of Genbank accession NC_009440 anddisclosed at SEQ ID NO:18, provided that the amino acid at residue 424is not the tryptophan present in a wild type Msed_1353. In oneembodiment, the amino acid at residue 424 is alanine, valine, leucine,isoleucine, or glycine. In one embodiment, the amino acid at residue 424is alanine, valine, leucine, glycine. In one embodiment, the amino acidat residue 424 is glycine. The amino acid sequence disclosed at SEQ IDNO:18 includes the substitution of glycine for tryptophan. Anotherexample is a polypeptide having structural similarity to the amino acidsequence SEQ ID NO:18, provided the amino acid at residue 424 is nottryptophan.

Additional examples of polypeptides expected to have4-hydroxybutyrate:CoA ligase activity include polypeptides catalyzing aCoA-ligase reaction that uses short (C2-C4) or medium (C5-C8) linearorganic acids as a substrate. For instance, examples of polypeptidesexpected to have 4-hydroxybutyrate:CoA ligase activity includepolypeptides catalyzing the reaction described under the IUBMB EnzymeNomenclature system as EC 6.2.1.1, EC 6.2.1.3, EC 6.2.1.17, or EC6.2.1.36. Such polypeptides may be obtained from members of the ordersDesulfurococcales (such as Ignicoccus hospitalis, or Pyrolobus fumarii),Thermoproteales (such as Thermoproteus neutrophilus), or Sulfolobales(such as Sulfolobus acidocaldarius, S. islandicus, S. solfataricus, S.tokodaii, Metallosphaera cuprina, or M. sedula).

A polypeptide having 4-hydroxybutyryl-CoA dehydratase activity means thepolypeptide catalyzes the conversion of 4-hydroxybutyryl-CoA tocrotonyl-CoA. The 4-hydroxybutyryl-CoA dehydratase activity of apolypeptide may be determined by routine methods known in the art. Anexample of such a polypeptide includes an amino acid sequence encoded bycoding sequence Msed_1321 of Genbank accession NC_009440 and disclosedat SEQ ID NO:15.

Other examples of polypeptides having 4-hydroxybutyryl-CoA dehydrataseactivity include a polypeptide having structural similarity to the aminoacid sequence encoded by coding sequence Msed_1321 of Genbank accessionNC_009440 and disclosed at SEQ ID NO:15.

Additional examples of polypeptides expected to have4-hydroxybutyryl-CoA dehydratase activity may be obtained from membersof the orders Sulfolobaceae (such as Metallosphaera sedula DSM 5348, M.yellowstonensis, M. prunae, and M. cuprina Ar-4, Acidianus hospitalisW1, Sulfolobus tokodaii str. 7, S. acidocaldarius DSM 639, S. islandicusY.G.57.14, S. islandicus Y.N.15.51, S. islandicus L.S.2.15, S.islandicus L.D.8.5, S. islandicus M.16.4, S. solfataricus P2, and S.islandicus M.14.25), and Desulfurococcales (such as Ignicoccushospitalis KIN4/I).

A polypeptide having crotonyl-CoA hydratase/(S)-3-hydroxybutyryl-CoAdehydrogenase activity means the polypeptide catalyzes the conversion ofcrotonyl-CoA to acetoacetyl-CoA. The crotonyl-CoAhydratase/(S)-3-hydroxybutyrl-CoA dehydrogenase activity of apolypeptide may be determined by routine methods known in the art. Anexample of such a polypeptide includes an amino acid sequence encoded bycoding sequence Msed_0399 of Genbank accession NC_009440 and disclosedat SEQ ID NO:16.

Other examples of polypeptides having crotonyl-CoAhydratase/(S)-3-hydroxybutyrl-CoA dehydrogenase activity include apolypeptide having structural similarity to the amino acid sequenceencoded by coding sequence Msed_0399 of Genbank accession NC_009440 anddisclosed at SEQ ID NO:16.

Additional examples of polypeptides expected to have crotonyl-CoAhydratase/(S)-3-hydroxybutyrl-CoA dehydrogenase activity may be obtainedfrom members of the orders Sulfolobaceae (such as Metallosphaera sedulaDSM 5348, M. yellowstonensis, M. prunae, and M. cuprina Ar-4, Acidianushospitalis W1, Sulfolobus tokodaii str. 7, S. acidocaldarius DSM 639, S.islandicus Y.G.57.14, S. islandicus Y.N.15.51, S. islandicus L.S.2.15,S. islandicus L.D.8.5, S. islandicus M.16.4, S. solfataricus P2, and S.islandicus M.14.25), Thermoproteales (such as Vulcanisaeta moutnovskia768-28 and V. distributa DSM 14429), Acidilobales (such as Acidilobussaccharovorans 345-15), and Desulfurococcales (such as Aeropyrum pernixK1, and Ignicoccus hospitalis KIN4/I).

A polypeptide having acetoacetyl-CoA β-ketothiolase activity means thepolypeptide catalyzes the conversion of acetoacetyl-CoA to acetyl-CoA.The acetoacetyl-CoA β-ketothiolase activity of a polypeptide may bedetermined by routine methods known in the art. An example of such apolypeptide includes an amino acid sequence encoded by coding sequenceMsed_0656 of Genbank accession NC_009440 and disclosed at SEQ ID NO:17.

Other examples of polypeptides having acetoacetyl-CoA β-ketothiolaseactivity include a polypeptide having structural similarity to the aminoacid sequence encoded by coding sequence Msed_0656 of Genbank accessionNC_009440 and disclosed at SEQ ID NO:17.

Additional examples of polypeptides expected to have acetoacetyl-CoAβ-ketothiolase dehydrogenase activity may be obtained from members ofthe orders Sulfolobaceae (such as Metallosphaera sedula DSM 5348, M.yellowstonensis, M. prunae, and M. cuprina Ar-4, Acidianus hospitalisW1, Sulfolobus tokodaii str. 7, S. acidocaldarius DSM 639, S. islandicusY.G.57.14, S. islandicus Y.N.15.51, S. islandicus L.S.2.15, S.islandicus L.D.8.5, S. islandicus M.16.4, S. solfataricus P2, and S.islandicus M.14.25), Thermoproteales (such as Vulcanisaeta moutnovskia768-28 and V. distributa DSM 14429), Acidilobales (such as Acidilobussaccharovorans 345-15), and Desulfurococcales (such as Aeropyrum pernixK1, and Ignicoccus hospitalis KIN4/I).

While this pathway is presented as a cycle, the skilled person willrecognize and appreciate that acetyl-CoA used by the heterotrimer E1αβγ,acetyl/propionyl-CoA carboxylase, does not need to originate from theenzymatic activity of E13 (acetoacetyl-CoA β-ketothiolase). Acetyl-CoAmay be produced through, for instance, the metabolism of amino acids,the degradation of fatty acids, or carbohydrate metabolism, andacetyl-CoA from any source may be the substrate of the heterotrimerE1αβγ.

A candidate polypeptide (e.g., a polypeptide having structuralsimilarity to a polypeptide described herein) may be isolated from amicrobe, such as an extremophile. An extremophile is an organism thatsurvives and thrives in challenging conditions impossible for mostorganisms. Examples of extremophiles include thermophiles,hyperthermophiles, acidophiles, and combinations thereof (e.g., athermoacidophile). “Thermophile” refers to prokaryotic microbes thatgrow in environments at temperatures of between 50° C. and no greaterthan 75° C. “Hyperthermophile” refers to prokaryotic microbes that growin environments at temperatures of at least 75° C. “Acidophile” refersto prokaryotic microbes that grow in environments at a pH of 3 or less.A prokaryotic microbe may be a member of the domain Archaea or a memberof the domain Bacteria. Examples of extremophiles include archaea suchas, but not limited to, members of the Order Thermococcales, members ofthe Order Sulfolobales, and members of the Order Thermotogales. Membersof the Order Thermococcales include, but are not limited to, a member ofthe genus Pyrococcus, for instance P. furiosus, P. abyssi, or P.horikoshii, a member of the genus Thermococcus, for instance, T.kodakaraensis or T. onnurineus. Members of the Order Sulfolobalesinclude, but are not limited to, a member of the genus Metallosphaera,for instance, M. sedula. Members of the Order Thermotogales include, butare not limited to, members of the genus Thermotoga, for instance, T.maritima or T. neapolitana. A candidate polypeptide may be producedusing recombinant techniques, or chemically or enzymaticallysynthesized.

The amino acid sequence of a polypeptide having structural similarity toa polypeptide described herein may include conservative substitutions ofamino acids present in an amino acid sequence. A conservativesubstitution is typically the substitution of one amino acid for anotherthat is a member of the same class. For example, it is well known in theart of protein biochemistry that an amino acid belonging to a groupingof amino acids having a particular size or characteristic (such ascharge, hydrophobicity, and/or hydrophilicity) may generally besubstituted for another amino acid without substantially altering thesecondary and/or tertiary structure of a polypeptide. For the purposesof this invention, conservative amino acid substitutions are defined toresult from exchange of amino acids residues from within one of thefollowing classes of residues: Class I: Gly, Ala, Val, Leu, and Ile(representing aliphatic side chains); Class II: Gly, Ala, Val, Leu, Ile,Ser, and Thr (representing aliphatic and aliphatic hydroxyl sidechains); Class III: Tyr, Ser, and Thr (representing hydroxyl sidechains); Class IV: Cys and Met (representing sulfur-containing sidechains); Class V: Glu, Asp, Asn and Gln (carboxyl or amidegroup-containing side chains); Class VI: His, Arg and Lys (representingbasic side chains); Class VII: Gly, Ala, Pro, Trp, Tyr, Ile, Val, Leu,Phe and Met (representing hydrophobic side chains); Class VIII: Phe,Trp, and Tyr (representing aromatic side chains); and Class IX: Asn andGln (representing amide side chains). The classes are not limited tonaturally occurring amino acids, but also include artificial aminoacids, such as beta or gamma amino acids and those containingnon-natural side chains, and/or other similar monomers such ashydroxyacids.

Guidance concerning how to make phenotypically silent amino acidsubstitutions is provided in Bowie et al. (1990, Science,247:1306-1310), wherein the authors indicate proteins are surprisinglytolerant of amino acid substitutions. For example, Bowie et al. disclosethat there are two main approaches for studying the tolerance of apolypeptide sequence to change. The first method relies on the processof evolution, in which mutations are either accepted or rejected bynatural selection. The second approach uses genetic engineering tointroduce amino acid changes at specific positions of a cloned gene andselects or screens to identify sequences that maintain functionality. Asstated by the authors, these studies have revealed that proteins aresurprisingly tolerant of amino acid substitutions. The authors furtherindicate which changes are likely to be permissive at a certain positionof the protein. For example, most buried amino acid residues requirenon-polar side chains, whereas few features of surface side chains aregenerally conserved. Other such phenotypically silent substitutions aredescribed in Bowie et al, and the references cited therein.

Guidance on how to modify the amino acid sequences of polypeptidesdisclosed herein is also provided at FIGS. 47-62. These figures show theamino acid sequences of polypeptides disclosed herein (SEQ ID NOs:1-17)in multiple protein alignments with other related polypeptides.Identical amino acids are marked with an asterisk (“*”), stronglyconserved amino acids are marked with a colon (“:”), and weaklyconserved amino acids are marked with a period (“.”). By reference tothese figures, the skilled person can predict which alterations to anamino acid sequence are likely to modify enzymatic activity, as well aswhich alterations are unlikely to modify enzymatic activity.

A polypeptide described herein may be expressed as a fusion polypeptidethat includes a polypeptide described herein and a heterologous aminoacid sequence. The heterologous amino acid sequence may be present atthe amino terminal end or the carboxy terminal end of a polypeptide, orit may be present within the amino acid sequence of the polypeptide. Forinstance, the heterologous amino acid sequence may be useful forpurification of the fusion polypeptide by affinity chromatography.Various methods are available for the addition of such affinitypurification tags to proteins. Examples of tags include apolyhistidine-tag, maltose-binding protein, and Strep-Tag®.Representative examples may be found in Hopp et al. (U.S. Pat. No.4,703,004), Hopp et al. (U.S. Pat. No. 4,782,137), Sgarlato (U.S. Pat.No. 5,935,824), Sharma (U.S. Pat. No. 5,594,115), and Skerra andSchmidt, 1999, Biomol Eng. 16:79-86). The heterologous amino acidsequence, for instance, a tag or a carrier, may also include a cleavablesite that permits removal of most or all of the additional amino acidsequence. Examples of cleavable sites are known to the skilled personand routinely used, and include, but are not limited to, a TEV proteaserecognition site. The number of heterologous amino acids may be, forinstance, at least 5, at least 10, at least 15, at least 20, at least25, at least 30, at least 35, or at least 40.

The polypeptides described herein may be produced by using recombinant,synthetic, or chemical techniques. For instance, a polypeptide may besynthesized in vitro, e.g., by solid phase peptide synthetic methods.Solid phase peptide synthetic methods are routine and known in the art.A polypeptide produced using recombinant techniques or by solid phasepeptide synthetic methods may be further purified by routine methods,such as fractionation on immunoaffinity or ion-exchange columns, ethanolprecipitation, reverse phase HPLC, chromatography on silica or on ananion-exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammoniumsulfate precipitation, gel filtration using, for example, Sephadex G-75,or ligand affinity. A preferred method for isolating and optionallypurifying a hydrogenase polypeptide described herein includes columnchromatography using, for instance, ion exchange chromatography, such asDEAE sepharose, hydrophobic interaction chromatography, such as phenylsepharose, or the combination thereof.

Also provided are isolated polynucleotides encoding the polypeptidesdescribed herein. For instance, a polynucleotide may have a nucleotidesequence encoding a polypeptide having the amino acid sequence shown inSEQ ID NOs:1-17, and an example of the class of nucleotide sequencesencoding each polypeptide is disclosed herein as a coding region ofGenbank accession NC_009440. It should be understood that apolynucleotide encoding a polypeptide represented by one of thesequences disclosed herein, e.g., SEQ ID NOs:1-17, is not limited to thenucleotide sequence disclosed as a coding region of Genbank accessionNC_009440, but also includes the class of polynucleotides encoding suchpolypeptides as a result of the degeneracy of the genetic code. Theclass of nucleotide sequences encoding a selected polypeptide sequenceis large but finite, and the nucleotide sequence of each member of theclass may be readily determined by one skilled in the art by referenceto the standard genetic code, wherein different nucleotide triplets(codons) are known to encode the same amino acid.

A polynucleotide disclosed herein can be present in a vector. A vectoris a replicating polynucleotide, such as a plasmid, phage, or cosmid, towhich another polynucleotide may be attached so as to bring about thereplication of the attached polynucleotide. Construction of vectorscontaining a polynucleotide of the invention may employ standardligation techniques known in the art. See, e.g., (Sambrook et al., 1989.Molecular cloning: a laboratory manual, 2nd ed. Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.). A vector can provide forfurther cloning (amplification of the polynucleotide), i.e., a cloningvector, or for expression of the polynucleotide, i.e., an expressionvector. The term vector includes, but is not limited to, plasmidvectors, viral vectors, cosmid vectors, and artificial chromosomevectors. Preferably the vector is a plasmid.

Selection of a vector depends upon a variety of desired characteristicsin the resulting construct, such as a selection marker, vectorreplication rate, and the like. Vectors can be introduced into a hostcell using methods that are known and used routinely by the skilledperson. The vector may replicate separately from the chromosome presentin the microbe, or the polynucleotide may be integrated into achromosome of the microbe. When more than one vector is to be used in acell, vectors having compatible origins of replication may be used(Adams et al. (US Patent Application 20110020875).

An expression vector may optionally include a promoter that results inexpression of an operably linked coding regino during growth inanaerobic conditions. Promoters act as regulatory signals that bind RNApolymerase in a cell to initiate transcription of a downstream (3′direction) coding region. The promoter is operably linked to a codingregion, and the coding region may encode an exogenous polypeptide or anendogenous polypeptide. In one embodiment, a promoter is operably linkedto more than one coding region, encoding exogenous polypeptides,endogenous polypeptides, or a combination thereof. Such an arrangementof one promoter controlling expression of two or more operably linkedcoding regions is often referred to as an operon. In one embodiment, aexogenous promoter may be present in the genomic DNA and operably linkedto an endogenous coding region.

In one embodiment, a suitable promoter causes expression of an operablylinked coding region at temperatures of at least 30° C., at least 40°C., at least 50° C., at least 60° C., at least 70° C., at least 80° C.,at least 90° C., or up at 100° C. In one embodiment, a suitable promotercauses expression of an operably linked coding region at temperaturesbetween 30° C. and 100° C., between 50° C. and 90° C., or between 60° C.and 80° C.

In one embodiment, a promoter is one that functions in an archaeon,e.g., a promoter that is recognized by a highly conserved transcriptioncomplex present in archaea cells. Archaeal promoters do not have thesame structure as promoters present in members of the domain Bacteria.One transcription factor important in the transcription of archaealcoding regions is TFB, a homologue of the eukaryotic TFIIB. Archaealpromoters often include a TATA box which may be centered 24 to 28nucleotides upstream of a transcription start site, and the TATA box canbe represented as a conserved 8 base pair sequence element TTTAWAta,where W is A or T, and R is A or G. An archaeal promoter may alsoinclude a TFB responsive element (cRNaANt), where R is A or G, and N isany nucleotide upstream and adjacent to the TATA box (Gregor andPfeifer, 2005, Microbiology, 151:25-33; Bell et al., 1999, Mol. Cell.,4:971-982; Bell et al., 1999, PNAS USA, 96:13662-13667).

In one embodiment, a promoter is one that functions in a member of thedomain Bacteria. The characteristics of bacterial promoters are known tothe person skilled in the art, and include, for instance, a −10 elementand a −35 element. A consensus sequence for the −10 element is TATAAT,and a consensus sequence for the −35 element is TTGACA; however, theseconsensus sequences are often not present in a promoter. Instead, a −10element and a −35 element of a bacterial promoter often has only threeor four of the six nucleotides in an element that match the consensus.Some bacterial promoters may also include an UP element, locatedupstream of the −35 element. Bacterial promoters are recognized bybacterial RNA polymerase, and are not recognized by a native RNApolymerase normally produced by an archaeon. Bacterial RNA polymeraseincludes 5 subunits, including a sigma subunit. Bacterial promotershaving a −10 element and a −35 element as described above are recognizedby an RNA polymerase that includes a sigma-70 subunit.

In those embodiments where a bacterial promoter is present in agenetically engineered archaeon, the genetically engineered archaeonrequires a bacterial RNA polymerase to drive expression of a codingregion operably linked to the bacterial promoter. Thus, a geneticallyengineered archaeon containing a bacterial promoter on an exogenouspolynucleotide also includes coding regions encoding the subunits of anRNA polymerase that will recognize and bind to a bacterial promoter andresult in expression of a coding region operably linked to the bacterialpromoter. A bacterial promoter and the coding regions encoding the RNApolymerase subunits may be on the same exogenous polynucleotide or maybe on separate exogenous polynucleotides in a genetically engineeredarchaeon. Coding regions encoding RNA polymerase subunits present on anexogenous polynucleotide present in a genetically engineered archaeonare operably linked to a promoter described herein, such as atemperature sensitive promoter or a constitutive promoter that functionsin an archaeon.

The promoter useful in methods described herein may be, but is notlimited to, a constitutive promoter, a temperature sensitive promoter, anon-regulated promoter, or an inducible promoter. A constitutivepromoter drives expression of an operably linked coding region in amicrobe when cultured at the temperatures described herein. Theexpression of a coding region operably linked to a constitutive promoteroccurs at both high and low incubation temperatures, and the level ofexpression does not change substantially when expression at higher andlower incubation temperatures is compared. An example of a constitutivepromoter is P_(slp), a P. furiosus promoter of the highly expressedS-layer protein (Chandrayan et al., 2012. J. Biol. Chem.,287:3257-3264). Other examples of constitutive promoters includeP_(gdh), P_(pep) and P_(porγ), which are promoters in both P. furiosusand T. kodakarensis of the highly expressed glutamate dehydrogenase,phosphoenolpyruvate synthase and pyruvate ferredoxin oxidoredutasesubunit γ, respectively (for example, see Lipscomb et al. 2011. Appl.Environ. Microbiol. 77:2232-2238; Chandrayan et al., 2012. J. Biol.Chem., 287:3257-3264).

The promoter may be a temperature sensitive promoter. In one embodiment,a temperature sensitive promoter drives expression of an operably linkedcoding region in a microbe at a greater level during incubation at lowtemperatures when compared to expression during incubation at hightemperature. Such a promoter is referred to herein as a “cold shock”promoter. A cold shock promoter is induced at temperatures lower thanthe optimum growth temperature (T_(opt)) of a microbe. In oneembodiment, a cold shock promoter is induced when a microbe is culturedat a temperature of no greater than 75° C., no greater than 70° C., nogreater than 65° C., no greater than 60° C., no greater than 55° C., nogreater than 50° C., no greater than 45° C., no greater than 40° C., orno greater than 35° C. In one embodiment, a cold shock promoter isinduced when a microbe is cultured at a temperature between 35° C. and45° C., between 40° C. and 50° C., between 45° C. and 55° C., between50° C. and 60° C., between 55° C. and 65° C., between 60° C. and 70° C.,or between 65° C. and 75° C. Induction of a cold shock promoter in agenetically engineered microbe may result in an upregulation ofexpression of an operably linked coding region by at least 10-fold, atleast 15-fold, at least 20-fold, at least 25-fold, or at least 30-foldcompared to expression of the same operably linked coding region duringgrowth of the genetically engineered microbe at its T_(opt).

Examples of cold shock promoters include those operably linked to thecoding regions of P. furiosus described by Weinberg et al., (2005, J.Bacteriol., 187:336-348). A promoter is present in the regionimmediately upstream of the first codon of a coding region. In oneembodiment, at least 150 nucleotides upstream to at least 200nucleotides upstream of the first codon of the operably linked codingregion includes the promoter. The size of the region that includes apromoter may be limited by the presence of an upstream coding regionsuch as a start codon (for a coding region on the opposite strand) or astop codon (for a coding region on the same strand). Identifyingpromoters in microbes, including hyperthermophilic archaeae andthermophilic archaeae, is routine (see, for example, Lipscomb et al.,2009, Mol. Microbiol., 71:332-349). Other archaea contain homologues ofthe coding regions described by Weinberg et al., and the promoters ofsuch homologues can be evaluated for induced expression at lowertemperatures. Cold sock promoters may be produced using recombinanttechniques.

In one embodiment, a temperature sensitive promoter drives expression ofan operably linked coding region in a microbe at a decreased levelduring incubation at low temperatures when compared to expression duringincubation at high temperature. Such a promoter is referred to herein asa “cold repressed” promoter. As described herein, a geneticallyengineered microbe may be used to produce a product; however, themicrobe may normally produce an endogenous enzyme that uses the productor an intermediate leading to the product. The use of a cold repressedpromoter is advantageous in such an embodiment. The geneticallyengineered microbe may be modified to decrease the production of theendogenous enzyme. For instance, a microbe may be genetically engineeredby removing the promoter driving expression of an endogenous enzyme andreplacing it with a cold repressed promoter.

A cold repressed promoter is repressed at temperatures lower than theT_(opt) of a microbe. In one embodiment, a cold repressed promoter isrepressed when a microbe is cultured at a temperature of no greater than75° C., no greater than 70° C., no greater than 65° C., no greater than60° C., no greater than 55° C., no greater than 50° C., no greater than45° C., no greater than 40° C., or no greater than 35° C. In oneembodiment, a cold repressed promoter is induced when a microbe iscultured at a temperature between 35° C. and 45° C., between 40° C. and50° C., between 45° C. and 55° C., between 50° C. and 60° C., between55° C. and 65° C., between 60° C. and 70° C., or between 65° C. and 75°C. The use of a cold repressed promoter in a genetically engineeredmicrobe may result in an down-regulation of expression of an operablylinked coding region by at least 10-fold, at least 15-fold, at least20-fold, at least 25-fold, or at least 30-fold compared to expression ofthe same operably linked coding region during growth of the geneticallyengineered microbe at its T_(opt).

Cold repressed promoters present in hyperthermophilic archaea andthermophilic archaea can be easily identified using routine methods. Forinstance, DNA microarray analysis can be used to compare expression ofcoding regions in an archaeon, such as a hyperthermophile, grown at itsT_(opt) and the arhaeon hyperthermophile grown at a temperature belowthe T_(opt). The temperature below the T_(opt) may be, for instance, atleast 20° C., at least 30° C., at least 40° C. below the T_(opt). Thedecrease in expression may be a change of at least 5-fold, at least10-fold, at least 15-fold, or at least 20-fold when comparing expressionat the two temperatures. Examples of cold repressed promoters include,but are not limited to, the promoter upstream of the hypotheticalpolypeptide encoded by coding region PF0882 of P. furiosus, the promoterupstream of the polypeptide encoded by coding region PF0421 of P.furiosus, and the promoter upstream of the polypeptide encoded by codingregion PF0198 of P. furiosus (Kelly et al., WO 2013/067326). Thepromoters of Kelly et al. may be used by attaching a coding region suchthat the first codon of the coding region is present immediatelyadjacent to and downstream of the nucleotide located at the 3′ end. Inone embodiment, a promoter includes at least 200 consecutivenucleotides, at least 250 consecutive nucleotides, at least 300consecutive nucleotides, at least 350 consecutive nucleotides, or atleast 400 consecutive nucleotides.

A vector may include a ribosome binding site (RBS) and a start site(e.g., the codon ATG) to initiate translation of the transcribed messageto produce the polypeptide. Like other regulatory sequences, a RBS maybe heterologous with respect to a host cell. When expressing anexogenous polynucleotide in P. furiosus, it was found that the RBSneeded to be carefully considered to ensure expression. A consensus RBSthat may be used in P. furiosus is TAGTGGAGGATA (SEQ ID NO:306), wherethe underlined portion of the consensus RBS is usually at nucleotideposition −10 to −5 relative to the start codon of the operably linkedcoding region. Other examples of useful RBS sequences include, but arenot limited to, GGTGATATGCAATG (SEQ ID NO:307), GGAGGTGGAGAAAATG (SEQ IDNO:308), GGAGGTTTGAAGATG (SEQ ID NO:309), GGAGGTGTGGGAAAATG (SEQ IDNO:310), and GGAGGGGGTGAGAGAGATG (SEQ ID NO:311), where the predictedRBS is underlined and the first codon of an operably linked codingregion is a double underlined ATG.

A vector may also include a termination sequence to end translation. Atermination sequence is typically a codon for which there exists nocorresponding aminoacetyl-tRNA, thus ending polypeptide synthesis. Thepolynucleotide used to transform the host cell can optionally furtherinclude a transcription termination sequence, and one example isAATCTTTTTTAG (SEQ ID NO:312).

A vector introduced into a host cell optionally includes one or moremarker sequences, which typically encode a molecule that inactivates orotherwise detects or is detected by a compound in the growth medium. Forexample, the inclusion of a marker sequence may render the transformedcell resistant to an antibiotic, or it may confer compound-specificmetabolism on the transformed cell. Examples of a marker sequenceinclude, but are not limited to, sequences that confer resistance tokanamycin, ampicillin, chloramphenicol, tetracycline, streptomycin, andneomycin. Examples of nutritional markers useful with certain hostcells, including extremophiles, are disclosed in Lipscomb et al. (USPublished Patent Application 20120135411), and include, but are notlimited to, a requirement for uracil, histidine, or agmatine.

Polynucleotides of the present invention may be obtained from microbes,or produced in vitro or in vivo. For instance, methods for in vitrosynthesis include, but are not limited to, chemical synthesis with aconventional DNA/RNA synthesizer. Commercial suppliers of syntheticpolynucleotides and reagents for such synthesis are well known.

Also disclosed herein are genetically engineered microbes that haveexogenous polynucleotides encoding one or more of the polypeptidesdisclosed herein. Compared to a control microbe that is not geneticallymodified in the same way, a genetically engineered microbe exhibitsproduction of 3-HP, 4-HB, acetyl-CoA, or another product, or exhibitsincreased production of 3-HP, 4-HB, acetyl-CoA, or another product.Accordingly, in one aspect of the invention a genetically engineeredmicrobe may include one or more exogenous polynucleotides that encodeone or more of the polypeptides described herein. Exogenouspolynucleotides encoding the polypeptides may be present in the microbeas a vector or integrated into a chromosome. In one embodiment, agenetically engineered microbe can exhibit an increase in production of3-HP, 4-HB, acetyl-CoA, or another product that at least 5%, 10%, 25%,50%, 75%, 100%, 150%, or 200% greater than the production of 3-HP, 4-HB,acetyl-CoA, or another product by an appropriate control.

Examples of useful bacterial host cells include, but are not limited to,Escherichia (such as Escherichia coli), Salmonella (such as Salmonellaenterica, Salmonella typhi, Salmonella typhimurium), a Thermotoga spp.(such as T. maritima), an Aquifex spp (such as A. aeolicus),photosynthetic organisms including cyanobacteria (e.g., a Synechococcusspp. such as Synechococcus sp. WH8102 or, e.g., a Synechocystis spp.such as Synechocystis PCC 6803) and photosynthetic bacteria (e.g., aRhodobacter spp. such as Rhodobacter sphaeroides), aCaldicellulosiruptor spp., such as C. bescii, and the like. Examples ofuseful archaeal host cells include, but are not limited to members ofthe Order Thermococcales (including a member of the genus Pyrococcus,for instance P. furiosus, P. abyssi, or P. horikoshii, or a member ofthe genus Thermococcus, for instance, T. kodakaraensis or T.onnurineus), members of the Order Sulfolobales (including a member ofthe genus Metallosphaera, for instance, M. sedula), and members of theOrder Thermotogales (including members of the genus Thermotoga, forinstance, T. maritima or T. neapolitana).

A genetically engineered microbe having exogenous polynucleotidesencoding one or more of the polypeptides disclosed herein optionallyincludes a source of electrons that can be used for the reduction of CO₂and/or other intermediates in the 4-HB cycle. In one embodiment, asource of electrons is hydrogenase, which catalyzes the reversibleinterconversion of H₂, protons, and electrons. A genetically engineeredmicrobe may naturally include a hydrogenase suitable for supplyingreductant, and in one embodiment, such a genetically engineered microbemay express an endogenous hydrogenase polypeptide at an increased levelor have altered activity. For instance, a genetically engineered microbemay include an altered regulatory sequence, where the altered regulatorysequence is operably linked to one or more coding regions encodingsubunits of a hydrogenase polypeptide. In another example, an endogenouspolynucleotide encoding a subunit of a hydrogenase polypeptide mayinclude a mutation, such as a deletion, an insertion, a transition, atransversion, or a combination thereof, that alters a characteristic ofthe hydrogenase polypeptides, such as the activity. In those aspectswhere a genetically engineered microbe expresses an endogenoushydrogenase polypeptide at an increased level or having alteredactivity, the microbe is typically an archaea, such as Pyrococcus spp.,such as P. furiosus, P. abyssi, and P. horikoshii, a Thermococcus spp.,such as T. kodakaraensis and T. onnurineus, and the like. Methods formodifying genomic DNA sequences of thermophiles and hyperthermophilesare known (Lipscomb et al. (US Published Patent Application20120135411).

In one embodiment, a genetically engineered microbe may includeexogenous polypeptides encoding the subunits of a hydrogenase. In oneembodiment, the hydrogenase may be an NADPH-dependent hydrogenase.Examples of hydrogenases and their expression in microbes are describedin Adams et al. (US Patent Application 20110020875), and Chandrayan etal. (2012, J. Biol. Chem., 287(5):3257-3264). In one embodiment, ahydrogenase includes 4 subunits, alpha, beta, gamma, and delta. In oneembodiment, a hydrogenase is 2 subunits, alpha and delta.

A genetically engineered microbe may include other modifications inaddition to exogenous polynucleotides encoding one or more of thepolypeptides disclosed herein, or expressing an endogenous hydrogenasepolypeptide at an increased level or having altered activity. Suchmodifications may provide for increased production of electron donorsused by a hydrogenase polypeptide, such as NADH or NADPH.

Also provided are methods for using the polypeptides described herein.In one embodiment, the methods include providing the polypeptides forsubpathway 1, subpathway 2, subpathway 3, or a combination thereof. Inone embodiment, a combination is subpathway 1 and subpathway 2. In oneembodiment, a combination is subpathway 1, subpathway 2, and subpathway3. In one embodiment, a combination is subpathway 2 and subpathway 3. Inone embodiment, a combination is subpathway 1 and subpathway 3. Thepolypeptides are incubated under conditions suitable for producingdesirable products such 3-HP, 4-HB, and/or other products. Optionally,the product is collected using methods routine and known in the art.

In one embodiment, a source of reductant is also provided. In oneembodiment, a source of reductant is provided by use of a hydrogenase.

In one aspect, the polypeptides used in the methods are cell-free. Forinstance, the polypeptides are isolated, or optionally purified. Theincubation conditions are typically anaerobic, and the temperature maybe at least 60° C., at least 70° C., at least 80° C., or at least 90° C.The methods can be performed in any convenient manner. Thus, thereaction steps may be performed in a single reaction vessel. The processmay be performed as a batch process or as a continuous process, withdesired product and waste products being removed continuously and newraw materials being introduced.

In another embodiment, the polypeptides used in the methods are presentin a genetically engineered microbial cell. The methods can includeincubating the microbial cell under conditions suitable for theexpression of the polypeptides. The microbial cell may be a bacterialcell, such as a gram negative, for instance, E. coli, a photosyntheticorganism, for instance, R. sphaeroides, or it can be an archaeal cell,for instance, a member of the genera Pyrococcus, for instance P.furiosus, P. abyssi, or P. horikoshii, or a member of the generaThermococcus, for instance, T. kodakaraensis or T. onnurineus. Theincubation conditions are typically anaerobic, and the temperature maybe at least 37° C., at least 60° C., at least 70° C., at least 80° C.,or at least 90° C. The use of these conditions results in severaladvantages. Growth at high temperatures reduces the risk ofcontamination, as growth of most microbes is reduced, or non-existent.The use of anaerobic conditions reduces the risks inherent in processingcompounds that can be used as fuels, such as combustion. Moreover, thehyperthermophiles like Pyrococcus and Thermococcus have genomes ofreduced complexity, and encode fewer polypeptides. The reducedcomplexity results in a more streamlined metabolism with fewerintermediates and decreased metabolic diversity. Hence, there is adecreased likelihood that there will overlap between the metabolitesand/or enzymes of the host with those in the engineered metabolicpathway.

The conditions used to incubate the microbial cell typically includesubstrates that can be used by a cell to produce a reductant, such asNADPH. In one embodiment, the conditions used to incubate the microbialcell can include H₂, which can be used by the hydrogenase polypeptide toconvert NADP to NADPH. The methods can be performed using any convenientmanner. For instance, methods for growing microbial cells to highdensities are routine and known in the art, and include batch andcontinuous fermentation processes.

In one embodiment, the method includes initial growth at a highertemperature followed by a shift to a lower temperature. The shift to alower temperature can result in greater activity of one or more of thepolypeptides described herein. In one embodiment, the greater activitymay be due to increased expression of a coding region encoding one ormore of the polypeptides, as is the case when a coding region isoperably linked to a temperature sensitive promoter. In one embodiment,the greater activity may be due to the shift to a temperature that isbetter tolerated by the one or more polypeptides. Further details onexpression of desired polypeptides below a microbe's T_(opt), and theproduction of desired products, are disclosed in Kelly et al. (WO2013/067326).

The methods disclosed herein may be used to make 3-HP, 4-HB, and otherproducts. The 4-HB cycle results in the production of acetyl CoA. AcetylCoA is an ideal product as it represents an activated reduced C-2 unitthat is of fundamental importance in conventional biosynthetic pathways.For example, acetyl CoA is the building block for the biosynthesis offatty acids, polyisoprenoids and hydroxyacids (such as 3-HB), all ofwhich are potential sources of alkane-based fuels and/or plastics. Thus,the 4-HB cycle can be used to directly generate a range of biofuels,including alkanes, biodiesel (fatty acid esters) and ethanol, as well asbutanol. Moreover, when converted to pyruvate, for instance by reductivecarboxylation, acetyl CoA can serve as the primary carbon and electronsource for all known biofuels (Connor et al., 2009, Curr Opin Biotechnol20:307-315, Lee et al., 2008, Curr Opin Biotechnol 19:556-63,Peralta-Yahya et al., Biotechnol J 5:147-62). Methods for convertingacetyl CoA to pyruvate are known and routine. Likewise, methods forconverting any compound produced by the 3-HP/4-HB cycle to other usefulproducts are known and routine. Other products that may be producedusing the methods disclosed herein include, but are not limited to,1,4-butanediol, succinic acid, isopropanol, ethanol, diols, and organicacids such as lactic acid, acetic acid, formic acid, citric acid, oxalicacid, and uric acid. The synthesis of 3-HP, 4-HB, acetyl-CoA, and otherproducts may be a starting material for the synthesis of othercompounds.

A method for using a genetically engineered microbe may also includerecovery of the product produced by the genetically engineered microbe.The method used for recovery depends upon the product, and methods forrecovering products resulting from microbial pathways, includingcarbohydrate metabolism, are known to the skilled person and usedroutinely. For instance, when the product is ethanol, the ethanol may bedistilled using conventional methods. For example, after fermentationthe product, e.g., ethanol, may be separated from the fermented slurry.The slurry may be distilled to extract the ethanol, or the ethanol maybe extracted from the fermented slurry by micro or membrane filtrationtechniques.

Also provided herein are methods for making a genetically engineeredmicrobe. The method includes introducing into a microbe at least onepolynucleotide. In one embodiment, the polynucleotide encodes apolypeptide described herein, so that the microbe produces 3-HP, 4-HB,acetyl-CoA, or another product. In one embodiment, the introducedpolynucleotide modifies an endogenous polynucleotide such thatexpression of an endogenous polypeptide is increased, or the amino acidsequence of an endogenous polypeptide is altered. An example of alteringthe amino acid sequence of an endogenous polypeptide includes modifyingthe amino acid sequence encoded by coding sequence Msed_1353 in a M.sedula such that the amino acid at residue 424 is not the tryptophanpresent in a wild type Msed_1353, and is a different amino acid, such asglycine.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

Example 1

Metallosphaera sedula is an extremely thermoacidophilic archaeon thatgrows heterotrophically on peptides, and chemolithoautotrophically onhydrogen, sulfur, or reduced metals as energy sources. Duringautotrophic growth, CO₂ is incorporated into cellular carbon via the3-hydroxypropionate/4-hydroxybutyrate cycle (3HP/4HB). To date, allsteps in the pathway have been connected to enzymes encoded in specificgenes, except for the one responsible for ligation of coenzyme A (CoA)to 4-hydroxybutyrate (4HB). While several candidates for this step havebeen identified through bioinformatic analysis of the M. sedula genome,none have been shown to catalyze this biotransformation. Here,transcriptomic analysis of cells grown under strict H₂—CO₂ autotrophyuncovered two additional candidates, encoded in Msed_0406 and Msed_0394.Recombinant versions of these enzymes catalyzed the ligation of CoA to4HB, with similar affinities for 4HB (Km values of 1.9 and 1.5 mM forMsed_0406 and Msed_0394, respectively), but with different rates (1.69and 0.22 μmol×min×mg⁻¹ for Msed_0406 and Msed_0394, respectively).Neither Msed_0406 nor Msed_0394 have close homologs in otherSulfolobales, although low sequence similarity is not unusual foracyl-adenylate forming enzymes. The capacity for these two enzymes touse 4HB as a substrate may have arisen from simple modifications toacyl-adenylate forming enzymes. For example, a single-amino acidsubstitution (Trp424 to Gly) in the active site of theacetate/propionate synthetase (Msed_1353), an enzyme that is highlyconserved among the Sulfolobales, changed its substrate specificity toinclude 4HB. The identification of the 4-HB CoA synthetase now completesthe set of enzymes comprising the 3HP/4HB cycle.

Experimental Procedures

Growth of M. sedula in a Gas Intensive Bioreactor

M. sedula (DSMZ 5348) was grown aerobically at 70° C. in a shaking oilbath (90 rpm) under autotrophic or heterotrophic conditions on DSMZmedium 88 at pH 2. Heterotrophically-grown cells were supplemented with0.1% tryptone. Cell growth was scaled up from 300 ml in sealed one literbottles (Auernik and Kelly, 2010, Appl. Environ. Microbiol. 76, 931-935)to 2 liters in a stirred bench-top glass fermentor (Applikon), also onDSMZ medium 88 (pH 2) at 70° C., and agitated at 250 rpm. Two separatelyregulated gas feeds were used such that flow rates were held constantfor all conditions at 1 ml/min for the hydrogen/CO₂ gas mixes(composition varied) and 100 ml/min for air (composition—78% N₂, 21% O₂,0.03% CO₂). For the autotrophic, carbon-rich (ACR) condition, the gasfeed contained H₂ (80%) and CO₂ (20%); for the autotrophiccarbon-limited (ACL) condition the feed was changed to H₂ (80%) and N₂(20%); for the heterotrophic condition (HTR), the medium wassupplemented with 0.1% tryptone and the gas feed composition was N₂(80%) and CO₂ (20%). Tandem fermentors were run simultaneously with thesame inoculum to generate biological repeats (FIG. 2). Cells wereharvested at mid-exponential phase by rapid cooling with dry ice andethanol, and then centrifuged at 6,000×g for 15 min at 4° C.

M. sedula Oligonucleotide Microarray Transcriptional Response Analysis

A spotted whole-genome oligonucleotide microarray, based on 2,256protein-coding open reading frames (ORFs), was used, as describedpreviously (Auernik and Kelly, 2008, Appl. Environ. Microbiol. 74,7723-7732). Total RNA was extracted and purified (RNeasy; Qiagen),reverse transcribed (Superscript III; Invitrogen), re-purified, labeledwith either Cy3 or Cy5 dye (GE Healthcare), and hybridized to themicroarray slides (Corning). Slides were scanned on a GenePix 4000BMicroarray Scanner (Molecular Devices, Sunnyvale, Calif.), and rawintensities were quantitated using GenePix Pro v6.0. Normalization ofdata and statistical analysis were performed using JMP Genomics 5 (SAS,Cary, N.C.). In general, significant differential transcription wasdefined to be relative change at or above 2 (where a log₂ value of ±1equals a two-fold change) with significance values at or above theBonferroni correction; for these data, this was 5.4 (equivalent to ap-value of 4.0×10⁶). Microarray data are available through the NCBI GeneExpression Omnibus (GEO) under accession number GSE39944.

Enzyme Assays for 4-Hydroxybutyrate-CoA Synthetase

Two assays were used to measure ligase activity, one spectrophotometricand one using high-performance liquid chromatography (HPLC). Adiscontinuous assay was used to measure substrate-dependentdisappearance of CoA at 75° C. The reaction mixture (600 μl) contained100 mM MOPS-KOH (pH 7.9), 5 mM MgCl₂, 2.5 mM ATP, 0.15 mM CoA, andpurified enzyme. At each time point, 80 μl of reaction mixture was addedto 80 μl cold 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB). A time point(0 min) was taken before heating. The reaction mixture was incubated for2 min at 75° C., followed by addition of substrate. Additional timepoints were taken at 30, 60, 90, 120, and 180 sec after addition ofsubstrate. Absorbance was measured at 412 nm to determine free CoAconcentration, based on the concentration of 2-nitro-5-thiobenzoatedianion (NTB²⁻) (ε₄₁₂=14,150 M⁻¹ cm⁻¹) (Hawkins et al., 2011, ACS Catal.1, 1043-1050, Riddles et al., 1983, Methods in Enzymology 91, 49-60).Enzymes were kinetically characterized by varying the concentration ofthe acyl-CoA substrate from 0.05 mM to 12 mM, while the other substrateconcentrations were held constant. Measurements for specific activitywere taken under saturating substrate concentrations (10 mM). Formationof the CoA ester was also confirmed using HPLC (Waters). The reactionmixture (0.15 ml) contained 100 mM potassium phosphate (pH 7.9), 10 mMMgCl₂, 2 mM ATP, 0.5 mM CoA, 10 mM substrate, and purified enzyme. Thereaction was incubated for 3 min at 75° C., quenched with 15 μl 1M HCl,filtered with a 10 kDa spin column (Amicon YM-10) to remove the protein,and loaded onto a reversed-phase C18 silica-based column (Shodex C18-4E,4.6×250 mm). The mobile phase was 50 mM sodium phosphate buffer (pH 6.7)with 2% methanol.

Heterologous Expression of M. sedula Genes in E. coli

M. sedula genes encoding acyl-CoA synthetases were amplified fromgenomic DNA using primers synthesized by Integrated DNA Technologies(Coralville, Iowa). Msed_0394 and Msed_0406 were ligated intopET46-Ek/LIC, while Msed_1353 was ligated into pET21b using NdeI andXhoI restrictions sites. All constructs were designed to express with anN-terminal His₆-tag. Plasmids containing gene inserts were cloned intoNovablue GigaSingles E. coli competent cells and selected by growth onLB-agar supplemented with ampicillin (100 μg/ml). Plasmid DNA wasextracted using a QIAprep Spin Miniprep kit. Sequences were confirmed byEton Biosciences, Inc. (Durham, N.C.). For protein expression, theplasmids were transformed into E. coli Rosetta 2 (DE3) cells andselected by growth on LB-agar, supplemented with ampicillin (100 μg/ml)and chloramphenicol (50 μg/ml). Cells harboring the recombinant plasmidwere induced with IPTG (final concentration 0.1 mM) at OD₆₀₀ 0.4-0.6 andcultured for three hours before harvest.

Purification of Recombinant Proteins

Cells were harvested by centrifugation at 6,000×g for 15 min at 4° C.Cell yields ranged from 1.6-3.8 g cells per liter LB medium (wetweight). Cell pellets were re-suspended in lysis buffer (50 mM sodiumphosphate, 100 mM NaCl, 0.1% NP-40, pH 8.0) containing DNase andlysozyme at final concentrations of 10 and 100 μg/ml, respectively.Cells were lysed with a French Press (two passes at 18,000 psi) and thelysate was centrifuged at 22,000×g for 15 min at 4° C. to removedinsoluble material. Soluble, cell-free extract was heated to 65° C. for20 min to precipitate mesophilic proteins. Streptomycin sulfate (1% w/v)was added to precipitate nucleic acids, followed by a one hourincubation at 4° C. A final centrifugation was performed at 22,000×g for15 min at 4° C. to collect the soluble, heat-treated cell-free extract,which was sterile filtered (0.22 μm) and purified using a 5 ml HisTrap™nickel column (GE Healthcare). Proteins were bound to the HisTrap™column using binding buffer (50 mM sodium phosphate, 500 mM NaCl, 20 mMimidazole, pH 7.4) and eluted using elution buffer (50 mM sodiumphosphate, 500 mM NaCl, 300 mM imidazole, pH 7.4). SDS-PAGE was thenperformed on the IMAC fractions to qualitatively determine the purity ofthe protein before further purification. Chromatography fractionscontaining the protein were concentrated and exchanged into phosphatebuffer (50 mM potassium phosphate, 150 mM NaCl, pH 7.0) using an AmiconYM10 (Millipore) centrifugal filter membrane, centrifuged at 4000×g and4° C. To quantify the amount of protein, a Bradford assay was performedon the concentrated IMAC fractions using known serial dilutions ofbovine serum albumin (BSA) by taking absorbance readings at 595 nm.Protein was further purified using a Superdex 200 10/300 GL (GEHealthcare) gel filtration column. The proteins were eluted from the gelfiltration column using elution buffer (50 mM potassium phosphate, 150mM NaCl, pH 7.0). Proteins were dialyzed into 100 mM MOPS-KOH (pH 7.9)and either stored at 4° C. or mixed with glycerol to 20% and stored at−20° C.

Site-Directed Mutagenesis of Msed_1353

Msed_1353 was mutated with the GENEART® Site-directed mutagenesis system(Life Technologies), using AccuPrime™ Pfx polymerase. Mutagenesisprimers were designed to change W424 to glycine (Primer1-5′-CCCTTTGGTAGCACTTGGGGAATGACTGAAACTGG (SEQ ID NO:312; Primer2—reverse compliment of Primer 1). Plasmids with Msed_1353-G424 werecloned into Novablue GigaSingles E. coli competent cells and selected bygrowth on LB-agar supplemented with ampicillin (100 μg/ml). Sequenceswere confirmed by Eton Biosciences Inc (Durham, N.C.).

Structural Modeling of Acyl-CoA Synthetases

Three-dimensional structural models for M. sedula acyl-CoA synthetaseswere made using the iterative threading assembly refinement (I-TASSER)online server (Berg 2011, Appl. Environ. Microbiol. 77, 1925-1936, Berget al., 2010, Nat. Rev. Microbiol. 8, 447-460, Roy et al., 2010, NatProtoc 5, 725-738). The server first generates three-dimensional atomicmodels from multiple threading alignments and iterative structuralassembly, and then infers function by structural matching to other knownproteins. All structures were generated using the Protein Data Baseentry for S. enterica Acs (STM4275, 1PG4) as a threading template foradditional restraint specification. Amino acid sequence alignments weregenerated using the UCSF Chimera package by superposition of I-TASSER 3Dstructural models with the PDB structure for S. enterica Acs.

Materials

Plasmid vectors and strains were obtained from Novagen (San Diego,Calif.) and Stratagene (La Jolla, Calif.). Chemicals, devices, andreagents were obtained from Fisher Scientific (Pittsburgh, Pa.), ACROSOrganics (Geel, Belgium), Sigma Chemical Co. (St. Louis, Mo.), NewEngland Biolabs (Ipswich, Mass.), Qiagen (Valencia, Calif.), Millipore(Billerica, Mass.) and Invitrogen (Grand Island, N.Y.). Gases werepurchased from Airgas National Welders (Charlotte, N.C.). Proteinpurification columns were obtained from GE Healthcare (Piscataway,N.J.). The Bradford Assay reagent was obtained from Bio-Rad (Hercules,Calif.). Site-directed mutagenesis kit was obtained from Invitrogen(Life Technologies).

Results

Metallosphaera sedula Autotrophic Growth is Hydrogen-Limited

In order to explore the optimal growth conditions for H₂—CO₂ autotrophyin M. sedula, a fermentation system was designed to allow controlleddefinition of the gas feed. Previous autotrophic work with M. sedula wasdone in batch cultures in an orbital shaking bath at 70° C. (Berg, 2011,Appl. Environ. Microbiol. 77:1925-1936, Berg et al., 2007, Science,318:1782-1786, Alber et al., 2008, J. Bacteriol. 190:1383-1389, Hugleret al., 2003, Arch. Microbiol. 179:160-173, Auernik and Kelly, 2010,Appl. Environ. Microbiol., 76:931-935). In that case, gas-fed cultureswere grown by replacing the air in a sealed volume with a gaseousmixture of a known composition. Mass transfer of H₂, CO₂, and O₂ intothe culture medium was limited to diffusion across the vapor-liquidinterface. Gas limitation presumably affected these cultures, and led tosub-optimal growth, as evidenced by the slow doubling time that resultedfor M. sedula under these conditions (t_(d)=11 to 13 h).

In order to grow M. sedula autotrophically with more optimal delivery ofgaseous substrate to the liquid medium, a semi-continuous fermentationsystem was developed using a 3 L bioreactor. The system was modified tohave two separate gas feeds that sparged directly into the media(sparging stone—2 μm pore size). Microbubble sparging stones were usedto promote dissolution of sparingly soluble gases, in particular H₂. Thebioreactor and console were situated inside a modified fume hood, withan airflow monitoring system in place to detect hood failure. Tandemfermentors were seeded with the same inoculum and run simultaneously toprovide a biological repeat.

Growth of M. sedula in an aerobic, autotrophic fermentation system wasexpected to be H₂-, and not O₂-limited. Below saturating conditions,growth rates varied according to the amount of H₂ fed to the culture.For high H₂ supply rates (i.e., 30 ml/min), the growth rates werecomparable to the fastest growth rates previously observed underheterotrophy (t_(d)=4.8 h); concomitantly, the culture reached a celldensity of 2×10⁹ cells/ml. the highest observed under autotrophicconditions. At a H₂ supply rate of 15 ml/min, the growth rate slowed(t_(d)=6 h) although the final density was comparable to the 30 ml/mincase (1.5×10⁹ cells/ml). A 30-fold reduction in H₂ flow rates (1 ml/min)caused the growth rate to decrease by half (t_(d)=9.7 h) and the cellsto enter stationary phase at 8×10⁸ cells/ml.

A similar trend emerged in response to limiting levels of CO₂. When CO₂was supplemented in the gas feed (referred to here as “rich”autotrophy), the growth rate was faster that observed for cells grownwith air as the only source of CO₂ (t_(d)=6.8 h vs. 9.4 h,respectively). The growth rate for heterotrophically grown cells(t_(d)=6.7 h) was comparable to the “rich” autotrophy condition. Thissuggests that, under the “rich” autotrophy condition, the cells were notlimited by any one particular gaseous substrate and were doubling at ornear their maximal rate. The decrease in growth rate for thecarbon-limited autotrophy arises from the limiting amounts of CO₂available in the medium.

Optimized H₂—CO₂ Autotrophy Conditions LED to Enhanced TranscriptomicResponse

The optimized autotrophic growth conditions enhanced the globaltranscriptional response compared to previous work (Berg et al., 2007,Science, 318:1782-1786, Huber et al., 2008, Proceedings of the NationalAcademy of Sciences, U.S.A, 105:7851-7856, Auernik and Kelly, 2010,Appl. Environ. Microbiol., 76:931-935). Of the 2293 protein coding genesin the 2.2 kb M. sedula genome, nearly half (984 genes) exhibitedchanges in transcription (either up- or down-regulation) of two-fold orgreater, when comparing heterotrophy (HTR) to the autotrophiccarbon-limited (ACL) condition (See Table 2). The number of genes thatwere differentially transcribed was twice as high as previously observed(Berg et al., 2010, Nat. Rev. Microbiol. 8:447-460, Auernik and Kelly,2010, Appl. Environ. Microbiol., 76:931-935), which could be attributedto the refined conditions for autotrophic growth. Also, in theexperiments reported here, it should be mentioned that the improvedsensitivity of new equipment used for scanning microarray slidesimproved the resolution and dynamic response.

TABLE 2 Enhanced Transcription Response for M. sedula Autotrophy ACL-ACRACL-HTR ACR-HTR A-H (1) # of genes UP-regulated 52 467 433 229 (2-foldor more) # of genes DOWN- 124 517 464 252 regulated (2-fold or more) (1)Auernik and Kelly, 2010, Appl. Environ. Microbiol. 76: 931-935

Overall, the global transcriptional changes were extensive. Transcriptsfor the characteristic enzymes of the 3HP/4HB pathway were significantlyup-regulated on ACL-HTR. For example, the genes encoding α- andβ-subunits of acetyl-CoA/propionyl-CoA carboxylase (Msed_0147-0148),were up-regulated 18- and 29-fold, respectively, while the4-hydroxybutyryl-CoA dehydratase gene (Msed_1321), was up-regulated27-fold. Hydrogenases and hydrogenase assembly and maturation proteinsin both the cytosolic hydrogenase operon (Msed_0921-0933) and themembrane-bound hydrogenase operon (Msed_0947-0950) were both highlyup-regulated on ACL-HTR, from 3- to 47-fold higher.

New Candidates for 4-Hydroxybutyrate-CoA Synthetase Identified fromRefined Transcriptomic Data

The refined transcriptomic data provided new insights into the putativecandidates for 4-hydroxybutyrate-CoA synthetase (FIG. 3). Based onbioinformatic analysis, there are nine candidate genes encoding acyl-CoAsynthetases (not including Msed_1456, which was confirmed as a 3HP-CoAsynthetase). The high up-regulation of Msed_1422 under autotrophy(13-fold increase) that was observed in this work is consistent withprevious transcriptomic studies. On the basis of that initial study,Msed_1422 was chosen for recombinant expression and testing (Berg, 2011,Appl. Environ. Microbiol. 77:1925-1936, Ramos-Vera et al., 2011, J.Bacteriol. 193:1201-1211, Estelmann et al., 2011, J. Bacteriol.193:1191-1200). In the same study recombinant forms of Msed_1291 andMsed_1353 were also produced, which were chosen based on homology to aconfirmed 4HB-CoA synthetase from Thermoproteus neutrophilus(Tneu_0420). None of these enzymes showed activity on 4HB. Msed_1422 andMsed_1291 showed no activity on acetate, propionate, 3HP, 3HB, 4HB, orcrotonate, and Msed_1353 had activity only on acetate and propionate,but not 4HB. Thus, it appears that Msed_1353 is a promiscuousacetate/propionate synthetase, while the substrate specificities ofMsed_1422 and Msed_1291 remain unknown.

Among the other potential candidates that were annotated as acetate-CoAsynthetases or medium-chain fatty acid-CoA synthetases (FIG. 3), mostshowed no transcriptional response, had average or low levels oftranscription, or were clearly down-regulated under autotrophy. The newtranscriptomic data were consistent with the expression of twopreviously unexamined candidates, Msed_0406 and Msed_0394, which areannotated as an acetyl-CoA synthetase (ACS) and AMP-dependent synthetaseand ligase, respectively. Although Msed_0406 and Msed_0394 were bothconstitutively transcribed, with less than a two-fold change intranscription levels between the conditions tested, both of them were inthe top 25% of the transcriptome. This served as the basis toinvestigate these two genes by recombinant expression and activityassays, given that no other promising candidates for this step hademerged.

Kinetic Analyses of Msed_0394 and Msed_0406

Recombinant forms of Msed_0394 and Msed_0406 were produced in E. coliand purified to electrophoretic homogeneity (see FIG. 9 for SDS-PAGEgels). For both enzymes, the production of 4HB-CoA from 4HB and CoA wasconfirmed using reversed-phase HPLC. Msed_0394 and Msed_0406 were activeon a range of small organic acids (see Table 3 for a summary of kineticdata). FIG. 4 shows the relative specific activities on differentsubstrates for Msed_0394, Msed_0406, along with reported data for3HP-CoA synthetase (Msed_1456) for comparison (Berg et al., 2007,Science, 318:1782-1786, Alber et al., 2008, J. Bacteriol. 190:1383-1389,Estelmann et al., 2011, J. Bacteriol. 193:1191-1200. Note that thecalculated molecular weight for these three enzymes varies slightly—62kDa for Msed_0394, 64 kDa for Msed_0406, and 74 kDa for Msed_1456; thesespecific activities here are meant to highlight substrate preferencepatterns for each enzyme.

TABLE 3 Enzyme kinetic data for CoA synthetases from M. sedula V_(max)(μmol min⁻¹ k_(cat) k_(cat)/K_(m) Enzyme Substrate K_(m) (μM) mg⁻¹)(s⁻¹) (s⁻¹ M⁻¹) Msed_0394 Acetate 680 0.13 0.14 200 Propionate 540 0.20.21 390 3- 1880 0.07 0.08 40 Hydroxypropionate 4-Hydroxybutyrate 15400.22 0.24 160 Butyrate 60 0.21 0.23 3700 Valerate 120 0.2 0.22 2000Msed_0406 Acetate 2030 6.0 6.4 3200 Propionate 380 15.1 16.2 43000 3-810 2.4 2.6 3200 Hydroxypropionate 4-Hydroxybutyrate 2000 1.7 1.8 910Butyrate 320 7.9 8.4 26000 Valerate 740 5.2 5.6 7500 Msed_1353-4-Hydroxybutyrate 1130 2.3 2.5 2180 G424

The specific activities for Msed_0394 show little difference in themaximum reaction rate under saturating substrate concentrations for thedifferent substrates. The highest reaction rate observed was ˜0.2 μmolmin⁻¹ mg⁻¹ for propionate, 4HB, and butyrate. However if the substratespecificities are taken into account a different picture emerges. Acomparison of the catalytic specificity constants (k_(cat)/K_(m)) foreach substrate tested with Msed_0394 (Table 3) shows that the highestvalue is for butyrate (3700 M⁻¹ s⁻¹), followed by valerate (2000 M⁻¹s⁻¹), propionate (390 M⁻¹ s⁻¹), acetate (200 M⁻¹ s⁻¹), and finally 4HB(160 M⁻¹ s⁻¹). There is a clear preference for unsubstituted straightchain organic acids with chain length of four or five carbons. Noactivity was detected with the six carbon hexanoic acid.

The specific activities for Msed_0406 under saturating substrateconcentrations show the highest reaction rates for propionate (15.1 μmolmin⁻¹ mg⁻¹). The catalytic specificity constant profile for Msed_0406shows that this enzyme works best on propionate (43000 M⁻¹ s⁻¹), thenbutyrate (26000 M⁻¹ s⁻¹), valerate (7500 M⁻¹ s⁻¹), acetate/3HP (3200 M⁻¹s⁻¹), and then 4HB (910 M⁻¹ s⁻¹). The high V_(max) foracetate/propionate, combined with the low K_(m) for propionate, suggestthat Msed_0406 is also a promiscuous acetate/propionate ligase, althoughone that also shows activity on 4HB.

Site-Directed Mutagenesis of Msed_1353

Msed_1353, a highly conserved gene among the Sulfolobales, waspreviously reported to have activity only on acetate and propionate(Berg et al., 2007, Science, 318:1782-1786, Alber et al., 2008, J.Bacteriol. 190:1383-1389, Ramos-Vera et al., 2011, J. Bacteriol.193:1201-1211, Hügler et al., 2003, Eur. J. Biochem. 270:736-744, Alberet al., 2006, J. Bacteriol. 188:8551-8559, Auernik et al., 2008, Appl.Environ. Microbiol. 74:7723-7732). Initial efforts to identify theunknown 4HB-CoA synthetase in M. sedula involved purification of nativeenzyme activity and analysis of multiple SDS-PAGE gel bands using massspectrometry. Msed_1353 was detected in these experiments and, based onthe very large up-regulation of Msed_1353 under autotrophy, it wasrecombinantly produced to confirm its activity. Our results confirmedprevious reports: under saturating substrate concentrations Msed_1353had highest activity on acetate (8.9 μmol min⁻¹ mg⁻¹-100%) andpropionate (99%), but also on 3HP (8%) and butyrate (16%). However, noactivity was found on 4HB or longer organic acid substrates (see FIG.5A).

Structural modeling of the binding pocket of Msed_1353 revealed aconserved tryptophan residue, similar to that seen in acetate-CoAsynthetase (ACS) from S. enterica (Berg et al., 2007, Science,318:1782-1786, Riddles et al., 1983, Methods in Enzymology 91:49-60,Gulick et al., 2003, Biochemistry 42:2866-2873). This tryptophan formsthe bottom surface of the binding pocket and limits the size ofsubstrate that can be accommodated within the active site. To test theimportance of this residue in determining substrate specificity, Trp⁴²⁴in Msed_1353 was mutated to a glycine to produce Msed_1353-G424. Thesingle substitution mutant (Trp⁴²⁴→Gly) was predicted to contain alarger interior binding pocket for the hydrophobic end of the substrate.Accordingly, it showed a dramatic change in specificity (FIG. 5B).Activity for the mutant on acetate and propionate decreased by 60%, from8.9 to 3.6 and 8.8 to 3.5 μmol min⁻¹ mg⁻¹, respectively. However,Msed_1353-G424 also showed activity on C4-C8 substrates, including 4HB(1.8 μmol min⁻¹ mg⁻¹).

In order to compare the activity of these three enzymes on 4HB theMichaelis-Menten curves are shown in FIG. 6. From this figure it isclear that there is a large difference in catalytic rate for the threeenzymes, and this difference holds over the entire range of substrateconcentration, including when [S]/K_(m)<<1. Therefore although it ispossible that both Msed_0394 and Msed_0406 are catalytically active on4HB in vivo, it is likely that Msed_0406 is more physiologicallyrelevant in terms of catalytic performance. Additionally, the singlepoint mutation of Msed_1353 to Msed_1353-G424 produces an enzyme that isactive on 4HB at even higher rates for all substrate concentrations.

DISCUSSION

The semi-continuous gas-intensive bioreactor system developed here wassuccessfully used to refine the transcriptional response ofautotrophy-related genes in M. sedula. This system provided betterdelivery of sparingly soluble gases and allowed more precise regulationof gas composition than could be achieved in serum bottles. At 70° C.and 1 atm, the solubility of oxygen and hydrogen are comparable (0.6mM), while the solubility of carbon dioxide is about 20-fold higher (12mM) (Auernik and Kelly, 2010, Appl. Environ. Microbiol., 76:931-935,Ramos-Vera et al., 2011, J. Bacteriol., 193:1201-1211, Wilhelm et al.,1977, Chem. Rev., 77:219-262). For these experiments, the low solubilityof H₂ was offset by the use of microbubbler sparing stones (2 μm poresize) to increase the gas phase surface area and increase delivery of H₂to the medium.

Stoichiometrically, at least four H₂ molecules are required for everycarbon atom fixed. Assuming that ATP generation requires the oxidationof two hydrogen molecules, then each turn of the cycle requires 12molecules of hydrogen for every two molecules of carbon dioxide. Assuch, the limiting growth factor for M. sedula in a bioreactor is likelyacquisition of the electron donor, in contrast to most aerobic microbialfermentation where acquisition of the final electron acceptor, oxygen,limits growth. In its natural environment, the picture may be somewhatdifferent. Hydrogen measurements from the (largely anoxic) acidic hotsprings at Yellowstone indicate that gaseous hydrogen may be quiteabundant—with concentrations ranging between 10-300 nM (Auernik andKelly, 2010, Appl. Environ. Microbiol., 76:931-935, Spear et al., 2005,Proc. Natl. Acad. Sci. U.S.A. 102:2555-2560). The source of thishydrogen gas is primarily geochemical; although the mechanism is notwell understood, it probably arises from subsurface interaction of waterwith Fe[II] (Auernik et al., 2008, Appl. Environ. Microbiol.74:7723-7732, Sleep, 2004, Proc. Natl. Acad. Sci. U.S.A.101:12818-12823). For most subsurface environments, oxygen is probablylimiting (Gold, 1992, Proc. Natl. Acad. Sci. U.S.A. 89:6045-6049).However, M. sedula was isolated from aerobic (surface) samples of a hotwater pond at Pisciarelli Solfatara (Huber et al., 1989, Syst. Appl.Microbiol. 12:38-47). Thus both hydrogen and oxygen may be available inabundance for autotrophic growth.

The regulation of growth modes in M. sedula involves massivetranscriptional changes between heterotrophic and autotrophic growth.Nearly half the genome (984 genes out of 2293) responded withtranscriptional changes of 2-fold or greater when comparing heterotrophyto carbon dioxide limited autotrophy. Not much is known about theregulation strategies employed by archaea to control gene transcription,but between different forms of chemolithoautotrophy (reduced metals, H₂,etc.) and heterotrophy, M. sedula can utilize a broad range of metabolicsubstrates for growth.

The missing step in the 3HP/4HB pathway has been the acyl-CoA synthetasethat utilizes 4HB. Previous attempts to identify the gene that encodesthis enzyme were unsuccessful, and the candidate enzymes had no activityon 4HB (Ramos-Vera et al., 2011, J. Bacteriol. 193:1201-1211). In thiswork, two previously unexamined synthetases from M. sedula, consistentwith the new transcriptomic evidence, were recombinantly produced andcharacterized. Both Msed_0394 and Msed_0406 showed activity on 4HB aswell as other small organic acids. Based on the lack of other synthetasecandidates suggested by the transcriptomic analysis and previousbiochemical evidence ruling out Msed_1422 and Msed_1291, we concludethat one or both of these enzymes are necessary for autotrophic growthin M. sedula.

Acetyl-CoA synthetases belong to the Class I superfamily ofadenylate-forming enzymes that includes acyl- and aryl-CoA synthetases,the adenylation domains of non-ribosomal peptide synthetases (NRPSs),and firefly luciferase (Schmelz and Naismith, 2009, Current Opinion inStructural Biology 19:666-671). These enzymes use a two-step mechanismin which first an acyl-AMP intermediate is formed (with release ofpyrophosphate) followed by displacement of AMP by CoA (Gulick, 2009, ACSchemical biology 4:811-827). Most acetyl-CoA synthetases have a limitedsubstrate range. Archaeal acyl-CoA synthetases, which form aphylogenetic cluster distinct from other bacterial subgroups (Brasen etal., 2005, Extremophiles 9:355-365), have been reported to exhibitbroader substrate preferences. The acetyl-CoA synthetase fromPyrobaculum aerophilum can work on acetate, propionate, butyrate, andisobutyrate (Bräsen et al., 2005, FEBS Lett. 579:477-482); anotheracetyl-CoA synthetase from Archaeoglobus fulgidus was active on acetate,propionate, and butyrate (Ingram-Smith and Smith, 2007, Archaea2:95-107). Both Msed_0394 and Msed_0406 were found to have activity on abroad range of small organic acid substrates of up to five carbons inlength.

Activity of both purified Msed_0394 and Msed_0406 on 4HB was well abovethe reported activity measured in autotrophic cell extract (0.3 μmolmin⁻¹ mg⁻¹) (Berg et al., 2007, Science, 318:1782-1786). It appears thatMsed_0406 is primarily a promiscuous propionate-CoA synthetase.Msed_0394, by contrast, has nearly equal levels of activity on acetate,propionate, and 4-HB. Although the overall activity for Msed_0394 islower by comparison, when taking into account the different substratespecificities, this enzyme shows a preference for C5-C6 linearunsubstituted organic acids. By comparison, the homologous 4-HB-CoAsynthetase from Thermoproteus neutrophilus (Tneu_0420), an anaerobicarchaeon that contains the DC/4HB carbon fixation cycle, wasrecombinantly produced and shown to have maximal activity on 4HB,followed by crotonate, acetate, 3HP, and 3HB (Ramos-Vera et al., 2011,J. Bacteriol. 193:1201-1211). The reported Km for Tneu_0420 is about3-fold lower than that found for Msed_0406 (700 μM vs. 2000 μM), withcomparable activity (1.6 vs. 1.8 μmol min⁻¹ mg⁻¹), which suggests thatthe catalytic activities on 4HB are also comparable.

It is likely that Msed_0406 is more effective at catalyzing the ligationof CoA to 4HB in vivo than Msed_0394. Perhaps these enzymes have evolvedfrom highly specific acetate/propionate synthetases to be sufficient forcatalyzing the necessary reaction on 4HB for the 3HP/4HB fixation cycle.It is not clear why two synthetases would be required, or if both ofthem are necessary for autotrophic growth. However, they are so far theonly ligases in M. sedula that have been shown to activate 4HB with CoA.

Genes with high homology to Msed_0394 and Msed_0406 exist in the genomeof the closely related M. cuprina (67% and 73% amino acid identity,respectively), but it is less clear whether homologs exist in thegenomes of other Sulfolobales, such as the Sulfolobus and Acidianus spp.Members of the acyl-adenylate forming enzyme family may share littleidentity or similarity in amino acid sequence apart from a few highlyconserved core motifs (Ingram-Smith and Smith, 2007, Archaea 2:95-107).There are homologs of Msed_0406 in other species of Sulfolobales thathave 30-35% identity, and one homolog in S. acidocaldarius with 61%identity. But the effort to find the M. sedula 4HB-CoA synthetase hasshown that substrate specificity cannot be inferred from amino acidsequence homology alone. However, the low homology of the M. sedula4HB-CoA synthetase gene does stand out among all the other genes in the3HP/4HB cycle, which have distinct homologs in Sulfolobus spp. thatrange from 50-80% identity.

Since 4HB is a metabolite unique to butyrate metabolism (Pryde et al.,2002, FEMS Microbiol. Lett. 217:133-139), including γ-aminobutyratefermentation (Gerhardt et al., 2000, Arch. Microbiol. 174:189-199) andpolyhydroxyalkanoate production (Valentin et al., 1995, Eur. J. Biochem.227:43-60), it is unlikely to have any other role in crenarchaealmetabolism outside of carbon fixation. Recent work with metabolic fluxanalysis has shown there is another exit route for carbon flux from thecycle through succinyl-CoA to succinate (Estelmann et al., 2011, J.Bacteriol. 193:1191-1200). In this study the authors estimate thattwo-thirds of the cycle carbon flux passes to succinate via succinyl-CoAor succinic semialdehyde, while one-third of the cycle carbon fluxpasses through the latter part of the cycle (via 4HB) to regenerateacetyl-CoA. Of course, this flux distribution may be highly dependent ongrowth conditions and could shift more to the 4HB branch depending onsubstrate availability.

It is clear that all members of the Sulfolobales order have a homologfor 4hbd, and therefore should have a complete set of enzymes for carbonfixation. But, previous studies have been mixed as to which Sulfolobusspp. are capable of autotrophic growth. Early reports on Sulfolobusacidocaldarius isolates claimed that they could growchemolithoautotrophically on elemental sulfur (Brock et al., 1972, Arch.Microbiol. 84:54-68, Shivvers and Brock, 1973, J. Bacteriol.114:706-710). Subsequent reports claim that neither S. solfataricus norS. acidocaldarius can grow autotrophically on elemental sulfur alone(Grogan, 1989, J. Bacteriol. 171:6710-6719), although it is unclearwhether they simply lost the ability to grow chemolithoautotrophicallyor were selected from what were originally mixed cultures (Kletzin etal., 2004, J. Bioenergetics and Biomembranes 36:77-91). Recent reportshave shown autotrophic growth of S. metallicus on sulfur and S. tokodaiion both sulfur and iron (Bathe et al., 2007, Appl. Environ. Microbiol.73:2491-2497). The only other member of the Sulfolobales that has beenreported to grow on hydrogen is Acidianus ambivalens, a sulfur-reducingacidophile (Laska, 2003, Microbiol. 149:2357-2371). Genes encoding forhydrogenase and maturation enzymes with homology to M. sedulahydrogenase genes are present in one strain of S. islandicus (HVE10/4),but this is predicted to be involved in anaerobic fermentation (Guo etal., 2011, J. Bacteriol. 193:1672-1680). Clearly, some Sulfolobus spp.must have a functional carbon fixation pathway, but others seem topossess an incomplete or non-functional pathway. It may be that theCoA-activating ligase that can operate on 4HB is essential for completecycle function, and loss of 4HB-CoA synthetase activity renders thecarbon fixation cycle inoperable.

To investigate the issue of substrate specificity, de novo structuralpredictions of M. sedula acyl-CoA synthetases with crystal structureswere compared with other known synthetases, including acetyl-CoAsynthetase from both S. enterica (Gulick et al., 2003, Biochemistry42:2866-2873) and S. cerevisiae (Jogl and Tong, 2004, Biochemistry43:1425-1431), and 4-chlorobenzonate-CoA synthetase from Alcaligenes sp.(Gulick et al., 2004, Biochemistry 43:8670-8679). The structure for ACSfrom S. enterica revealed that there are four residues that form theacetate binding pocket—Val³¹⁰, Thr³¹¹, Val³⁸⁶, and Trp⁴¹⁴ (Gulick etal., 2003, Biochemistry 42:2866-2873). The conserved tryptophan residuecuts the binding pocket short and precludes activity on longersubstrates (FIG. 7). Extensive mutagenesis of binding pocket residues inyeast ACS showed that mutation of Trp⁴¹⁶→Gly⁴¹⁶ was sufficient tolengthen the binding pocket to accommodate C4-C8 organic acids(Ingram-Smith et al., 2006, Biochemistry 45:11482-11490). Amino acidsequence alignments show that Msed_1353 has a tryptophan in the sameposition (Trp⁴²⁴) (FIG. 8) and should, therefore, only work on acetateand propionate, a fact that has been confirmed biochemically (Ramos-Veraet al., 2011, J. Bacteriol. 193:1201-1211). Here, there was someactivity with Msed_1353 on 3HP and butyrate, but no activity on 4HB.Msed_0394 and Msed_0406 both have a glycine in this position, G333 andG346, respectively. However, the rest of the genes annotated as acyl-CoAsynthetases in M. sedula also have a glycine in this position, so thisglycine residue alone is not sufficient to indicate activity on C3-C5unsubstituted linear organic acids. Both Msed_1422 and Msed_1291 wererecombinantly expressed and showed to be inactive on C2-C4 linearorganic acids (Ramos-Vera et al., 2011, J. Bacteriol. 193:1201-1211).

A mutant of Msed_1353 with a glycine in place of the conservedtryptophan (Trp⁴²⁴→Gly) was made by site directed mutagenesis andexpressed in E. coli (Msed_1353-G424). The native enzyme was active onlyon acetate and propionate, but the mutant showed activity on 3HP, 4HB,valerate, hexanoate, and even octanoate (FIG. 5). The activity was justas high on C5-C8 substrates as on acetate and propionate, but lower on3HP and 4HB. This suggests that the polar hydroxyl group destabilizesthe interaction between the substrate and the residues of the enlargedbinding pocket. A similar trend is evident with Msed_0406 (FIG. 4).However, Msed_0394 has nearly equal levels of activity on propionate,butyrate, and 4HB, suggesting that it can stabilize the hydroxyl groupon 4HB better than that of 3HP. Similarly, Msed_1456, which catalyzesthe ligation of CoA to 3HP in the 3HP/4HB pathway, has equal activity onpropionate and 3HP, and therefore might have residues in the active sitethat help stabilize the hydroxyl group of 3HP.

In Msed_1456, Val³⁸⁶, which makes contacts with the γ-carbon of thepropyl moiety in the S. enterica ACS structure, is replaced with Asn³⁹⁰,whose polar amide nitrogen could hydrogen bond with the hydroxyl groupof 3HP to stabilize substrate binding. As for Msed_0406, both valineresidues in the acetate binding pocket are replaced with alanine (Ala²⁴⁹and Ala³²¹) and Thr³¹¹ is replaced with a lysine (Lys²⁵⁰). In Msed_0394,all three of these residues are alanine (Ala²⁴⁰, Ala²⁴¹, and Ala³⁰⁹).Potential candidate residues for stabilizing the hydroxyl group of 4HBin Msed_0394 include His³⁴¹ and Tyr³³⁸.

This work helps to close the gaps on the missing piece of the 3HP/4HBpathway in M. sedula. It is still unclear why only certain members ofthe Sulfolobales operate the 3HP-4HB cycle, but this may reflect theenvironmental history of specific species. Furthermore, along with otherrecent successes obtaining recombinant versions of difficult to produceenzymes from the pathway (Han et al., 2012, Appl. Environ. Microbiol.,78:6194-202), complete characterization of all cycle enzymes is near athand. The information obtained for cycle function will be invaluable forthe creation of a metabolically engineered platform capable of producingof chemicals and fuels from carbon dioxide (Hawkins et al., 2011, ACSCatal. 1:1043-1050).

Example 2 Production of an Industrial Chemical Using Hydrogen and CarbonDioxide

Microorganisms can be engineered to produce useful products, includingchemicals and fuels from sugars derived from renewable feedstocks, suchas plant biomass. An alternative method is to utilize low potentialreducing power from non-biomass sources, such as hydrogen gas orelectricity, to reduce carbon dioxide directly into products. Thisapproach circumvents the overall low efficiency of photosynthesis andthe production of sugar intermediates. While significant advances havebeen made in manipulating microorganisms to produce useful products fromorganic substrates, engineering them to utilize carbon dioxide andhydrogen gas has not been reported. Herein, we describe a noveltemperature-dependent approach that confers upon a microorganism, thearchaeon Pyrococcus furiosus, that grows optimally on carbohydrates at100° C., the capacity to utilize carbon dioxide, a reaction that it doesnot accomplish naturally. This was achieved by the heterologousexpression of five genes of the carbon fixation cycle of the archaeonMetallsphaera sedula, which grows autotrophically at 73° C. Theengineered P. furiosus strain is able to utilize hydrogen gas andincorporate carbon dioxide into 3-hydroxypropionic acid, one of the toptwelve industrial chemicals building blocks. The reaction can beaccomplished by cell-free extracts and by whole cells of the recombinantP. furiosus strain. Moreover, it is carried out some 30° C. below theoptimal growth temperature of the organism, conditions that support onlyminimal growth but maintain sufficient metabolic activity to sustain theproduction of 3-hydroxypropionate. The approach described here can beexpanded to produce important organic chemicals, all through biologicalactivation of carbon dioxide.

Materials and Methods

Construction of a synthetic SP1 operon. PCR was performed using P.furiosus or M. sedula genomic DNA to generate the individual PCRproducts of the P. furiosus S-layer promoter (P_(slp)) and the five M.sedula SP1 genes, consisting of coupled E1αβ (Msed_0147-Msed_0148), E1γ(Msed_1375), E2 (Msed_0709) and E3 (Msed_1993). P. furiosus ribosomalbinding sites, consisting of 11-14 bp of sequence upstream ofhighly-expressed proteins, were added in front of E1γ (5′-GGAGGTTTGAAG(SEQ ID NO:313), sequence upstream from porγ, PF0791), E2(5′-GGGAGGTGGAGCAT (SEQ ID NO:314), sequence upstream from slp, PF1399),and E3 (5′-GGTGATATGCA (SEQ ID NO:315), sequence upstream from cipA,PF0190). The primer sequences are given in Table 4. SOE-PCR (splicing byoverlap extension and PCR, (Horton et al., 1989, Gene 77(1):61-68) wasperformed to combine the individual PCR products and generate theexpression cassette for SP1 (FIG. 11A).

TABLE 4 Primers used in the construction of the synthetic SP1 operon.Primer target Direction 5′ to 3′ sequence P_(s/p) ForwardGAATCCCCGCGGCCCGGGCTGGCAGAATAGAA (SEQ ID NO: 316) ReverseGCAACCAAAACTCTACTAAAGGGTGGCATTTTTCTCCACCTCCCAATAATCTG (SEQ ID NO: 317)Msed_0147- Forward ATGCCACCCTTTAGTAGAGTTTTGG (SEQ ID NO: 318) 0148ReverseGTTGCAGTCATCTTCAAACCTCCTTACTTTATCACCACTAGGATATCTCC (SEQ ID NO: 319)Msed1375 ForwardGTGATAAAGTAAGGAGGTTTGAAGATGACTGCAACTTTTGAAAAACCGGAT (SEQ ID NO: 320)ReverseCGTTCTCCTCATATGCTCCACCTCCCTTAGAGGGGTATATTTCCATGCTTC (SEQ ID NO: 321)Msed_0709 ForwardGGCAATGTCATATGAGGAGAACGCTAAAGGCCGCAATTC SEQ ID NO: 403) ReverseCCTTTTCAGTCATTGCATATCACCTCATCTCTTGTCTATGTAGCCCTTC(SEQ ID NO: 322)Msed_1993 ForwardTAGACAAGAGATGAGGTGATATGCAATGACTGAAAAGGTATCTGTAGTTGGAG (SEQ ID NO: 323)Reverse CCAATGCATGCTTATTTTTCCCAAACTAGTTTGTATACCTTC (SEQ ID NO: 324)

Construction of vectors for insertion of the SP1 operon into P.furiosus. The SP1 expression cassette (FIG. 11B) was cloned into pSPF300(Hopkins et al., 2011, PLoS One 6(10):e26569), generating the plasmidpALM506-1, to be used for targeted insertion of the synthetic SP1 operoninto the P. furiosus ΔpdaD strain (FIG. 14). SOE-PCR (Horton et al.,1989, Gene 77(1):61-68) was used to combine ˜0.5 kb flanking regionstargeting homologous recombination in the integenic space betweenconvergent genes PF0574-PF0575, with a marker cassette, includingrestriction sites for cloning. The marker cassette for uracilprototrophic selection consisted of the pyrF gene driven the gdhpromoter region (Pgdh, 157 bases upstream of PF1602) and terminated with12 bases of the 3′ UTR of the hpyA1 gene (5′-aatcttttttag (SEQ IDNO:326), PF1722). A 65-b sequence of the 3′ end of the marker cassette(5′-ctaaaaaagattttatcttgagctccattctttcacctcctcgaaaatcttcttagcggcttccc(SEQ ID NO:327)) was repeated at the beginning of the cassette to serveas a homologous recombination region for selection of marker removal(Farkas et al., 2012, Appl Environ Microb 78(13):4669-4676). VectorpGL007 targeting homologous recombination at the PF0574-PF0575intergenic space was constructed by cloning the SOE-PCR product intopJHWOO6 (Lipscomb et al., 2011, Appl Environ Microb 77(7):2232-2238)(FIG. 15). The SP1 expression cassette was PCR-amplified from pALM506. Aterminator sequence was added to the 3′ end of the operon(5′-aatcttttttag (SEQ ID NO:328), from the 3′ UTR of PF1722), and theconstruct was cloned into the AscI-NotI sites of pGL007 to make pGL010(FIG. 16), for targeted insertion of the SP1 operon at the PF0574-PF0575intergenic space. Transformation of P. furiosus ΔpdaD strain wasperformed as previously described for COM1 (Lipscomb et al., 2011, ApplEnviron Microb 77(7):2232-2238) except that the defined medium containedmaltose instead of cellobiose as the carbon source and was supplementedwith 0.1% w/v casein hydrolysate. Transformation of COM1 was performedas previously described (Lipscomb et al., 2011, Appl Environ Microb77(7):2232-2238) except that linear plasmid DNA was used fortransformation.

Growth of P. furiosus. Strains were cultured as previously described ina sea-water based medium containing 5 g/L maltose and 5 g/L yeastextract, 0.5 g/L riboflavin, and 20 μM uracil or 4 mM agmatine as needed(Lipscomb et al., 2011, Appl Environ Microb 77(7):2232-2238). Cultureswere grown at 95° C. until ˜1×10⁸ cells/mL and then cooled at 23° C.until the temperature reached 70 to 75° C., which was maintained for upto 48 hours. For growth in a 20 L fermenter, the culture was spargedwith 10% CO₂/90% N₂, stirred, and the pH was maintained at 6.8 byaddition of 10% NaHCO₃. Cell extracts prepared anaerobically asdescribed previously (Lipscomb et al., 2011, Appl Environ Microb77(7):2232-2238) in 100 mM MOPS, pH 7.5, re-concentrated three-timeswith a 3 kDa centrifugation filter and stored at −80° C.

Growth of M. sedula for biochemical assays and product analysis. M.sedula (DSM 5348) was grown autotrophically at 70° C. withmicro-bubblers feeding 1 mL/min 80/20 H₂/CO₂ and 100 mL/min air in thedefined medium, DSMZ 88, at pH 2.0 as previously described (Han et al.,2012, Appl Environ Microbiol 78(17):6194-6202). To obtain cell-freeextracts, frozen cell pellets were anaerobically suspended in 50 mM TrisHCl pH 8.0 containing 0.5 μg/mL DNase I and stirred for 1 hr in ananaerobic chamber. The cell extract was centrifuged at 100,000×g for 1hr and the supernatant was stored at −80° C.

E1, E2 and E3 assays. All reactions were carried out in sealed anaerobiccuvettes at 75° C. containing 100 mM MOPS pH 7.5, 5 mM MgCl₂, 5 mM DTT.After addition of NADPH (to A₃₄₀˜1.0) and the relevant substrate (seebelow), NADPH oxidation was measured at 340 nm. The substrates for theE2, E2+E3 and E1+E2+E3 assays were succinyl-CoA, malonyl-CoA and acetylCoA (each 1 mM) respectively. The latter assay also contained 1 mM ATP,and 10 mM NaHCO₃. E1 activity was measured by phosphate release. Theassay contained 10 mM NaHCO₃, 1 mM ATP, and 1 mM acetyl-CoA. Samples (20μL) were removed at 2-4 min, diluted with water (180 μl), and theBioVision (Mountain View, Calif.) phosphate assay reagent (20 μl) wasadded. The phosphate produced was calculated using a molar extinctioncoefficient of 90,000 M⁻¹ cm⁻¹ at 650 nm.

Measurement of 3-hydroxypropionic acid (3-HP). 3-HP (H0297, 30%, w/v, inwater) was obtained from TCI America (http://www.tciamerica.net/). ByHPLC and ¹H NMR, it was 75% pure with the remaining 25% as3,3′-oxydipropanoic acid. For GC-MS analysis, inositol was the internalstandard. Samples were freeze-dried, incubated in 2 M trifluoroaceticacid at 80° C. for 1 hr, dried under nitrogen, andper-O-trimethylsilylated by treatment with Tri-Sil (Pierce) at 80° C.for 30 minutes. GC-MS analysis was performed on an AT 7890n GCinterfaced to a 5975C MSD using a Grace EC-1 column (30 m×0.25 mm). Theexact mass of 3-HP-TMS is 162. Derivatization of 3-HP with 2-nitrophenylhydrazine was carried out as described previously (Miwa et al., 2000,Journal of Chromatography. A 881(1-2):365-385). The 3HP-hydrazide wasextracted by adding 1.0 mL of 1 M KPO₄ buffer pH 7.0 and 1.5 mL of etherto 800 μL of the sample, centrifuging for 10 min at 6,000×g to separatethe phases, removing the top ether layer and evaporating. The driedsample was resuspended in 200 μL ethanol and 10-50 μL aliquots wereanalyzed by HPLC. The column and run conditions were as follows: column,Supelco LiChrosorb RP-8 (5 μm); solvent system, A 0.05% TFA, B 100%acetonitrile; gradient 0-20 min, 0-100% B, 20-22 min: 100% B; flow rate:1 mL/min; temperature: 30° C. For ESI-MS analysis, the dried derivativewas dissolved in methanol and directly injected on a Perkin-Elmer API 1plus in negative mode. The mass of the anionic 3-HP-hydrazide derivativeis 224.

Production of 3-HP in vitro from malonyl-CoA by E2+E3 and fromacetyl-CoA by E1+E2+E3. To the P. furiosus extract (1-2 mg/mL) in 100 mMMOPS pH 7.5, 5 mM MgCl₂, and 5 mM DTT, was added 1-2 mM malonyl-CoA (forE2+E3) or 10 mM NaHCO₃ (or 100% CO₂ in the gas phase), 2 mM ATP and 2 mMacetyl-CoA (for E1+E2+E3). The electron source was 2 mM NADPH or 0.5 mMNADP with 20% H₂ in the headspace. Sealed anaerobic vials containing thereaction mixture were incubated at 75° C. for up to 2 hr. Samples werederivatized with 2-nitrophenyl hydrazine and analyzed for 3-HP by HPLCas described above.

Product analysis of E1+E2+E3 activities in whole cells. P. furiosusstrains PF506 and MW56 were grown in 2 L cultures at 95° C. for 10 hoursuntil cell densities of 1×10⁸ cells/mL and then cooled and incubated at75° C. for 16 hours. Harvested cells were suspended to 5×10¹⁰ cells/mLin 100 mM MOPS pH 7.5 and base salts (28 g/L NaCl, 3.5 g/L MgSO₄.7H₂O,2.7 g/L MgCl₂.6H₂O, 0.33 g/L KCl, 0.25 g/L NH₄Cl, 0.14 g/L CaCl₂.2H₂O).The cell suspension was sealed in a serum vial, degassed with argon, andcysteine HCl (0.5 g/L), NaHCO₃ (10 mM) and either maltose (10 mM) orpyruvate (40 mM) were added. The vials were degassed and flushed with H₂and incubated at 75° C. for 60 minutes. Samples for 3-HP analysis werederivatized with 2-nitrophenyl hydrazine, using 1 mM p-hydroxyphenylacetic acid as an internal standard, ether-extracted and analyzed byHPLC as described above.

Analysis of the P. furiosus culture medium for 3-HP. P. furiosus strainsPF506, MW56 and COM1 were grown at 98° C. in 50 mL cultures with maltose(10 mM) as the carbon source until a cell density of 8×10⁷ cells/mL wasreached. The incubation temperature was then shifted to 72° C. for up to4 days. Sample (1 mL) were periodically removed, centrifuged (10,000×g,10 min) and to a 100 μl aliquot of the supernatant (the spent medium) 1mM p-hydroxyphenyl acetic acid was added as an internal standard. Thesample was derivatized with 2-nitrophenyl hydrazine, ether-extracted andanalyzed by HPLC as described above.

Results and Discussion

The genes that were incorporated into P. furiosus to enable it toutilize carbon dioxide are the first part of the3-hydroxypropionate/4-hydroxybutyrate pathway of M. sedula, whichconsists of 13 enzymes in total (Ramos-Vera et al., 2011, J Bacteriol193(5): 1201-1211). In one turn of the cycle, two molecules of carbondioxide are added to one molecule of acetyl-CoA (C₂) to generate asecond molecule of acetyl-CoA (FIG. 11C). The cycle can be divided intothree sub-pathways (SP1-SP3) where SP1 generates 3-hydroxypropionate(3-HP) from acetyl-CoA and carbon dioxide, SP2 generates4-hydroxybutyrate (4-HB) from 3-HP and carbon dioxide, and SP3 converts4-HB to two molecules of acetyl-CoA. The reducing equivalents and energyfor the pathway are supplied by NADPH and ATP, respectively (FIG. 11D).Notably, the 3-HP/4-HB pathway is purportedly more energeticallyefficient than carbon dioxide fixation by the ubiquitous Calvin cycle(Berg et al., 2007, Science 318(5857):1782-1786).

The first three enzymes of the Msed 3-HP/4-HB cycle comprise the SP1pathway and together they produce 3-HP from carbon dioxide andacetyl-CoA (FIG. 11B). The three enzymes are referred to here as: E1(acetyl/propionyl-CoA carboxylase, encoded by Msed_0147, Msed_0148,Msed_1375), E2 (malonyl/succinyl-CoA reductase, Msed_0709) and E3(malonate semialdehyde reductase, Msed_1993) (Berg et al., 2007, Science318(5857):1782-1786; Hügler et al., 2003, Eur J Biochem 270(4):736-744;Alber et al., 2006, J Bacteriol 188(24):8551-8559). E1 carboxylatesacetyl-CoA using bicarbonate and requires ATP. E2 breaks theCoA-thioester bond and with E3, reduces the carboxylate to an alcoholwith NADPH as the electron donor. E1 and E2 are bi-functional and arealso involved in the SP2 part of the cycle (FIG. 11C). To demonstratethe concept, we expressed the M. sedula SP1 pathway in P. furiosus sothat the organism could utilize carbon dioxide for the production of3-HP, using hydrogen as the electron donor. Hydrogen is utilized in P.furiosus by a native cytoplasmic hydrogenase (SHI) that reduces NADP toNADPH (Ma & Adams, 2001, Method Enzymol Volume 331:208-216). SHI isextremely active, even at 70° C., and a P. furiosus strain engineered toover-express the enzyme was previously developed (Chandrayan et al.,2012, J Biol Chem 287(5):3257-3264).

The five genes encoding the three enzymes (E1αβγ, E2, E3) of M. sedulaSP1 were combined into a single synthetic operon with transcriptiondriven by P_(slp), a native, constitutive promoter of the highlyexpressed S-layer protein (PF1399) of P. furiosus (Chandrayan et al.,2012, J Biol Chem 287(5):3257-3264). The M. sedula ribosomal bindingsites (RBS) for E1(γ), E2 and E3 were replaced with RBSs for knownhighly-expressed P. furiosus proteins (FIG. 1A). The M. sedula RBS forE1β was retained since the two genes, E1α and E1β, appear to betranslationally-coupled. The SP1 operon was inserted into P. furiosus(strain COM1) at two genome locations. In P. furiosus strain PF506, theSP1 operon was inserted at the site of the pdaD marker (PF1623; FIG.14). The MW56 strain contained the SP1 operon betweenconvergently-transcribed genes (PF0574 and PF0575: FIGS. S2 and S3)within a ˜100-bp region having little to no transcriptional activity,according to a previous tiling array study of P. furiosus (Yoon et al.,2011, Genome Res 21(11): 1892-1904). The P. furiosus strains used hereare summarized in Table 5.

TABLE 5 Strains used and constructed in this study. Strain ParentGenotype/Description Reference COM 1 DSM ΔpyrF 1 3638 ΔpdaD COM 1 ΔpyrFΔpdaD::P_(gdh)pyrF 2 PF506 ΔpdaD ΔpyrF ΔpdaD::pdaD P_(slp) ⁻E1αβγ-E2-This work E3 MW56 COM 1 ΔpyrF P_(gdh)pyrF P_(slp) ⁻E1αβγ-E2-E3 This work1, Lipscomb et al., 2011, Appl Environ Microbiol 77: 2232-2238; 2,Hopkins et al., 2011, PLoS One 6: e26569.

The premise for the temperature-dependent strategy is that P. furiosus(T_(opt) 100° C.) shows little growth and has very low metabolicactivity (Weinberg et al., 2005, J Bacteriol 187:336-348) near thetemperature at which the enzymes from M. sedula (T_(opt) 73° C.) areexpected to be optimally active. In the recombinant P. furiosus strains(PF506 and MW56), the SP1 operon was under the control of atemperature-independent, constitutive promoter (P_(slp)), hence theoperon will be transcribed at both 100° C. and 75° C. However, theresulting E1−E3 enzymes should be stable and active only near 75° C. P.furiosus strains PF506 and MW56 were, therefore, grown at 98° C. (to˜1×10⁸ cells/ml) in closed static cultures and then transferred to 75°C. (FIG. 12A). There was no measurable activity of E1, E2 or E3 incell-free extracts prior to the temperature change, but all threeactivities were present in cells after 16 hr at 75° C. Moreover, thespecific activities were comparable to those measured in extracts of M.sedula cells grown autotrophically on hydrogen and carbon dioxide and tovalues reported by others (FIGS. 12C and 20) (Berg et al., 2010, Nat RevMicrobiol 8(6):447-460; Ramos-Vera et al., 2011, J Bacteriol 193(5):1201-1211). Indeed, when grown in a stirred, pH-controlled culture, theactivity of the linked E2+E3 enzymes in strain MW56 continued toincrease over a 50 hr period, reaching over 8-fold greater than thatmeasured in M. sedula (FIG. 13C). When strain PF506 was grown at 95° C.and then incubated for 16 hours at temperatures between 55° and 95° C.,the maximum specific activity of the linked E2+E3 enzymes was measuredin cultures incubated at 70 and 75° C., with dramatically lower valuesat 65 and 80° C. (FIG. 12B). This clearly indicates that the M. sedulaenzymes functioned optimally in P. furiosus at 70-75° C., especiallysince significant E2+E3 activity could be measured at assay temperaturesabove 75° C. using cell-free extracts prepared from cultures incubatedat 70-75° C. (FIG. 12D). Moreover, the enzymes are very thermostable,with a half-life of approximately 60 min at 90° C. (FIG. 18). Thissuggests that the lack of enzyme activity of the M. sedula enzymes (andof 3-HP production) in cultures that were incubated at 80° C. or higheris not due to the thermal instability of the M. sedula enzymes per se,but rather to the temperature sensitivity of the protein folding processduring the synthesis of these enzymes, which is optimal in the 70-75° C.range.

To determine the nature of the products of the SP1 pathway, recombinantP. furious strains PF506 and MW56 were grown at 95° C. (to ˜1×10⁸cells/ml) and then transferred to 70° C. for 16 hours (FIG. 19). Inextracts of these cells, the specific activities of the E1, E2, and E3enzymes were comparable to those measured in extracts ofautotrophically-grown M. sedula cells (FIG. 20). Two methods were usedto detect 3-HP and to confirm its production by the SP1 pathway in therecombinant P. furiosus strains. In the presence of acetyl-CoA, NaHCO₃,and either NADPH or hydrogen gas as the electron donor, the2-nitrophenylhydrazide-derivative (3-HP/HZ; m/z 224) was identified byelectrospray ionization mass spectrometry (ESI-MS) in cell-free extractsof PF506, but was not detected in extracts of the parent P. furiosusstrain (FIG. 21). This was confirmed by gas chromatography-massspectrometry (GC-MS) of the O-trimethylsilylate derivative of 3-HP(3HP/TMS), using malonyl-CoA and either NADPH or hydrogen gas as theelectron donor (Table 6). The GC-MS also allowed quantitation of3-HP/TMS and showed that approximately 150 μM 3-HP was produced frommalonyl-CoA, after a 2 hr incubation at 72° C. with extracts of PF506containing NADP under hydrogen gas (Table 6).

TABLE 6 3-HP/ Added Inositol Electron Theoretical peak Estimated VialDonor Substrate 3-HP area 3-HP 1 2 mM 2 mM 1 mM 0.0288 0.2 mM NADPHmalonyl-CoA 2 2 mM 2 mM 2 mM 0.0467 0.3 mM NADPH, H₂ malonyl-CoA 3 1 mM2 mM 2 mM 0.0274 0.2 mM NADP, H₂ malonyl-CoA 4 1 mM None 0 0.0064 0.05mM  NADP, H₂ (control) 5 1 mM None 2 mM 0.2839 2.0 mM NADP, H₂ (control)

For routine analysis of 3-HP, a method was developed to extract 3-HP/HZand to separate and quantitate it by HPLC. As shown in FIG. 3A, thismethod was used to confirm 3-HP production from acetyl-CoA and carbondioxide by the combined action of the enzymes E1, E2, and E3 incell-free extracts. As expected, P. furiosus did not appear to furthermetabolize 3-HP, as the compound was stable when added to P. furiosuscultures. Moreover, the production of 3-HP from acetyl-CoA was dependentupon either NaHCO₃ or CO₂ as the C-1 carbon source and either NADPH orhydrogen gas (and NADP) as the electron donor (FIG. 13A). Theincorporation of electrons from hydrogen gas and the carbon from carbondioxide into a single desired product is essentially the paradigm for‘electrofuels’ (Hawkins et al., 2011, ACS Catalysis 1:1043-1050).

P. furiosus grows by fermenting sugars (such as the disaccharidemaltose) to acetate, carbon dioxide and hydrogen, and can also utilizepyruvate as a carbon source (Fiala & Stetter, 1986, Arch. Microbiol.145: 56-61). Acetyl-CoA and carbon dioxide are generated as the productof the pyruvate ferredoxin oxidoreductase (POR) reaction (FIG. 22). Thereduced ferredoxin is oxidized by a membrane-bound hydrogenase togenerate hydrogen gas (Sapra et al., 2003, Proc Natl Acad Sci USA100(13):7545-7550). Although growth is limited at 75° C. (Weinberg etal., 2005, J Bacteriol 187:336-348), it was expected that when wholecells were incubated at 75° C. with maltose or pyruvate, sufficientacetyl-CoA would be produced by the low metabolic activity of P.furiosus for the SP1 enzymes to produce 3-HP. This was confirmed by HPLCdetection and quantitation of 3-HP as the 2-nitrophenylhydrazidederivative. For example, high cell density suspensions (≧10¹⁰ cells/ml)of P. furiosus strains PF506 and MW56 produced up to 0.2 mM 3-HP afterone hour incubation at 75° C. in the presence of maltose, hydrogen gas,and NaHCO₃ (FIG. 23), and 3-HP production was dependent upon thepresence of maltose or pyruvate (Table 7). Moreover, recombinant P.furiosus strains PF506 and MW56, grown in static cultures to late-logphase (˜1×10⁸ cells/ml) at 98° C. on maltose, produced up to 0.6 mM 3-HP(60 mg/1) when subsequently incubated at 72° C. for up to 40 hours (FIG.13B). Furthermore, in a stirred, pH-controlled culture, strain MW56produced 3-HP continuously over a 50 hr period at 72° C. (FIG. 13C).Overall, there appeared to be no significant difference between the tworecombinant P. furiosus strains in terms of 3-HP production. Thisindicated that the genome location of the synthetic operon derived fromM. sedula was not a determining factor. This bodes well for theinsertion of additional synthetic operons in P. furiosus to extend theresults reported here to other industrial chemicals.

TABLE 7 3-HP production using maltose or pyruvate as the source ofacetyl-CoA by whole cells of P. furiosus strains PF506 and MW56. Theamount of 3-HP indicated was present in 1 mL of the cell suspension ofP. furiosus. MW56 PF506 Pyruvate Maltose Pyruvate Maltose 155 nmol 100nmol 70 nmol 145 nmol

In summary, this work demonstrates the use of hydrogen as the electrondonor for carbon dioxide fixation into a product of great utility in thechemical industry, namely 3-HP. Moreover, it is carried out by anengineered heterotrophic hyperthermophile some 30° C. below the optimalgrowth temperature of the organism, conditions that support minimalgrowth, but sufficient metabolic activity is retained to sustain theproduction of 3-HP (Hawkins et al., 2011, ACS Catalysis 1:1043-1050).The reaction can be accomplished by cell-free extracts, and also bywhole cells in culture using sugar (maltose) as the source of theacetyl-CoA and ATP in a hydrogen- and carbon dioxide-dependent manner.The feasibility of using hydrogen gas as the source of reducing power(NADPH) for chemical synthesis, in this case 3-HP, is also of highsignificance given the availability of relatively inexpensive naturalgas as a hydrogen source (Kreysa, 2009, ChemSusChem 2(1):49-55). It isimportant to note that the low metabolic activity of P. furiosus at 72°C. was sufficient to provide the ATP needed for carbon dioxide fixation.These results are a significant step forward towards the overall goal ofincorporating into P. furiosus the complete M. sedula 3-HP/4-HB pathway,in which two molecules of carbon dioxide are reduced to acetyl-CoA thatcan then be converted into a variety of valuable products includingbiofuels (Hawkins et al., 2011, ACS Catalysis 1:1043-1050). Clearly,there will be a balance between using a fixed carbon source (sugar) viathe low metabolic activity of the host to produce ATP and the highcatalytic activity of the heterologous enzymes to generate the desiredproduct. The hydrogen-dependent fixation of carbon dioxide has enormouspotential for the production of a variety of chemicals and fuels throughstrategic use of established biosynthetic pathways and exploiting thehyperthermophilicity of metabolically-engineered microbial hosts (Steenet al., 2010, Nature 463(7280):559-562); Peralta-Yahya & Keasling, 2010,Biotechnol J 5(2): 147-162; Connor & Liao, 2009, Curr Opin Biotechnol20(3):307-315; Kreysa, 2009, ChemSusChem 2(1):49-55).

Example 3 Construction of P. furiosus Strains PF506 and MW56 Containingthe SP1 Pathway for 3-Hydroxypropionate Production and the ControlStrain MW43 for Optimizing Production of M. sedula Enzymes in P.furiosus

The five genes encoding the three enzymes (E1αβγ, E2, E3) of the M.sedula 3-HP/4-HB CO₂ fixation sub pathway I (SP1) are scattered acrossthe M. sedula genome (FIG. 24). These genes have been combined into asingle artificial operon using overlapping SOE-PCR (splicing by overlapextension and PCR, Horton, et al. 1989. Gene 77, 61), followed byintegration of the expression cassette into the P. furiosus genome.Transcription of the artificial SP1 operon in P. furiosus is driven byP_(slp), the native, constitutive promoter of the highly expressedS-layer protein (Chandrayan, S. K. et al. 2012. J. Biol. Chem. 287,3257-3264). To optimize translation of the SP1 genes in P. furiosus, thenative M. sedula ribosomal binding sites (RBSs) for E1γ, E2 and E3 werereplaced with optimal P. furiosus RBSs/linker regions for predicted andknown highly expressed proteins, while retaining the M. sedula RBS forE1β since the two genes, E1α and E1β, appear to be translationallycoupled.

Strategy for operon expression (SP1 and SP2B) in P. furiosus. The SP1operon was inserted into the COM1 strain of P. furiosus at two locationson the genome giving rise to two recombinant P. furiosus strains, PF506and MW56. In addition, a control strain, MW43, was constructed toexplore the temperature dependent expression of M. sedula genes in P.furiosus. MW43 contained subpathway 2B (SP2B; E7, E8 and E9) of the3HP/4HB cycle.

PF506: the SP1 operon was inserted at the site of the pdaD marker.

MW56: the SP1 operon was inserted into one (GR3) of eleven genomeregions previously identified as having little or no transcriptionalactivity.

MW43: the SP2B operon was inserted into GR2.

Construction of synthetic operon for expression of SP1 genes. PCR wasperformed using P. furiosus genomic DNA or M. sedula genomic DNA togenerate the individual PCR products of the P. furiosus S-layer promotorand the five M. sedula SP1 genes, consisting of coupled E1αβ(Msed_0147-Msed_0148), E1γ (Msed_1375), E2 (Msed_0709) and E3(Msed_1993). PCR primers were designed to contain optimized P. furiosusribosomal binding sites and spacing (Table 4) and to allow splicing ofthe individual PCR products generated (Table 4 and Table 8). SOE-PCR(Horton, et al. 1989. Gene 77, 61) was performed to combine theindividual PCR products and generate the expression cassette for SP-1(FIG. 25). The expression cassette was digested with SacII and SphIrestriction enzymes and cloned into the SacII-SphI sites of thetransformation vector, pSPF300 (Hopkins et al., 2011, PLoS One6(10):e26569), generating the transformation plasmid, pALM506-1, fortargeted insertion into the ΔpdaD strain of P. furiosus (FIG. 26).

TABLE 8Upstream and intergenic regions with optimized native Pf RBS sequences and spacing.E1-α: Msed_0147 GGGAGGTGGAGAAAATG (SEQ ID NO: 329)PF1399 (s/p, S-layer protein) RBS E1-β3: Msed_0148GGGTGATGTGGGGATGA (SEQ ID NO: 330)Msed0148 (native Msed RBS: coupled E1-αβ3) E1-γ: Msed_1375TAAGGAGGTTTGAAGATG (SEQ ID NO: 331) PF0791 (porγ: Pyruvate ferredoxinoxidoreductase γ) RBS E2: Msed_0709TAAGGGAGGTGGAGCATATG (SEQ ID NO: 332) PF1399 (s/p, S-layer protein) RBSE3: Msed_1993 TGAGGTGATATGCAATG (SEQ ID NO: 333)PF0190 (cipA, cold induced protein A) RBS)

Transformation of P. furiosus ΔpdaD strain to yield P. furiosus strainPF506 containing the SP1 operon. Transformation of P. furiosus ΔpdaDstrain was performed as previously described for COM1 (Lipscomb, et al.2011. Appl Environ Microbiol. 77(7):2232-8) with a few changes, in thatsequence-verified plasmid DNA was used for transformation and thedefined medium contained maltose instead of cellobiose as the carbonsource and was supplemented with 0.1% w/v casein hydrolysate. Briefly,pALM506-1 was mixed (at ˜5 μg plasmid DNA/mL culture) with an aliquot ofa fresh overnight culture of ΔpdaD grown in defined maltose (DM) mediumcontaining 0.1% w/v casein hydrolysate and 4 mM agmatine. Thetransformation mixtures were spread on DM plate medium containing 0.1%w/v casein hydrolysate and 20 μM uracil and incubated at 90° C. for ˜95h. Transformant colonies were further purified by six serial transfersin DM liquid medium containing 0.1% w/v casein hydrolysate and 20 μMuracil. The presence of the insert in the transformed strains wasverified by PCR screening of isolated genomic DNA.

Determining transcriptionally inactive regions for foreign geneinsertion. P. furiosus intergenic genome regions with little to notranscriptional activity were found using tiling array data of geneexpression in wild-type P. furiosus from early log to early stationaryphase, relative to a mid-log time point ((Yoon, et al. 2011. Genome Res.21(11):1892-904), FIG. 27). Primary targets consisted of intergenicspace between convergent genes, so as to avoid gene promoter regions.Secondary targets consisted of intergenic space between genes in thesame orientation, separated by at least ˜450 bases. Ten total genomeregions with little to no transcriptional activity were identified foruse as foreign gene insertion sites. Tiling array data was mapped to theNCBI reference genome sequence (P. furiosus DSM3638); however, thegenetically tractable strain of P. furiosus, COM1, has some genomerearrangements which affect the positions of the genome regions withinthe chromosome (Lipscomb G L, et al. 2011. Appl Environ Microbiol.77(7):2232-8, Bridger S L, et al. 2012. J Bacteriol. 194(15):4097-106)(FIG. 28). Namely, genome region 10 was located within a region of theP. furiosus genome which was inverted in the COM1 strain.

Construction of vectors targeting insertion at genome regions 2 and 3.SOE-PCR (splicing by overlap extension and PCR, Horton, et al. 1989) wasused to combine ˜0.5 kb flanking regions targeting homologousrecombination at genome region 3 (between convergent genesPF0574-PF0575, see FIG. 28), with a marker cassette, includingrestriction sites for cloning. The marker cassette for uracilprototrophic selection consisted of the pyrF gene driven by either thepep promoter region (P_(pep), 123 bases of DNA sequence immediatelyupstream from the translation start of the PEP synthase gene, PF0043) orthe gdh promoter region (P_(gdh), 157 bases of DNA sequence immediatelyupstream from the translation start of the glutamate dehydrogenase gene,PF1602) and terminated with the terminator sequence consisting of 12bases of the 3′ UTR of the hpyA1 gene (5′-aatcttttttag (SEQ ID NO:334),PF1722). A 65-b sequence of the 3′ end of the marker cassette(5′-ctaaaaaagattttatcttgagctccattctttcacctcctcgaaaatcttcttagcggcttccc(SEQ ID NO:335)) was repeated at the beginning of the cassette to serveas a homologous recombination region for selection of marker removalfrom the transformed strain which would allow for iterative use of themarker in the same strain (Farkas J, et al. Appl Environ Microbiol.2012. 78(13):4669-76) (FIG. 29). Vector pGL002, targeting genome region2, was constructed by cloning the SOE-PCR products into the SmaI site ofpJHW006 (FIG. 30), and vector pGL007 targeting genome region 3 wasconstructed by cloning the SOE-PCR product into the NdeI-NheI sites ofpJHW006 (FIG. 31) (Lipscomb, et al., Appl Environ Microb 77:2232-2238(2011)).

Construction of synthetic operons (SP1 and SP2B) for expression of Msedgenes in P. furiosus. SOE-PCR was used to construct artificial operonsfor the co-expression of SP2B genes consisting of the four M. sedulagenes E7 (Msed_0639), E8α (Msed_0638), E8β (Msed_2055), E9 (Msed1424),with expression driven by the slp promoter region (P_(slp), consistingof 184 bases immediately upstream from the slp gene, PF1399). P.furiosus ribosomal binding sites from either the pep gene(5′-ggaggtttgaag (SEQ ID NO:336)) or the slp gene (PF1399,5′-ggaggtggagaaaa (SEQ ID NO: 337)) were inserted in front of each genedownstream from the first in the operon. A terminator sequence of thehpyA1 gene was included at the end of the operon (5′-aatcttttttag (SEQID NO:338), from the 3′ UTR of PF1722) (FIG. 32). The SP2B operonconstruct was cloned into the SmaI site of pGL002 to make pGL005 fortargeted insertion at P. furiosus genome region 2 (FIG. 33).

The expression cassette for SP1 consisting of the five M. sedula genesE1α (Msed_0147), E1β (Msed_0148), E1γ (Msed_0149), E2 (Msed_0709), E3(Msed_1993) was PCR-amplified from pALM506 (FIG. 34). This expressioncassette contained ribosomal binding sites from the PORγ gene (PF0791,5′-ggaggtttgaag (SEQ ID NO:339)), the slp gene (PF1399,5′-ggaggtggagaaaa (SEQ ID NO:340)), and the cipA gene (PF0190,5′-ggtgatatgca (SEQ ID NO:341)). A terminator sequence was added to the3′ end of the operon (5′-aatcttttttag (SEQ ID NO:342), from the 3′ UTRof PF1722), and the construct was cloned into the AscI-NotI sites ofpGL007 to make pGL010 (FIG. 35), for targeted insertion at P. furiosusgenome region 3 (see FIG. 27).

Transformation of P. furiosus COM1 strain to yield P. furiosus strainMW56 containing SP1 and strain MW43 containing SP2B. Transformation ofCOM1 was performed as previously described (Lipscomb, et al., ApplEnviron Microb 77:2232-2238 (2011)), except that linear plasmid DNA wasused for transformation. Briefly, pGL010 and pGL005 were linearized byrestriction digest and mixed (at a final concentration of ˜2 μg/mL DNA)with an aliquot of a freshly grown culture of COM1, cultured in definedcellobiose medium plus 20 M uracil. Transformation mixtures were spreadon defined cellobiose plate medium without uracil and incubated at 95°C. for ˜60 h. Transformant colonies were further purified on definedcellobiose plate medium without uracil twice. Strains were verified byPCR screening of isolated genomic DNA and sequencing of PCR productsamplified from the target regions.

Example 4 Temperature-Dependent Production of M. sedula Enzymes in P.furiosus Using Strains PF506 (E1−E3) and MW43 (E9)

Growth of P. furiosus for biochemical assays and product analysis. P.furiosus strains were cultured in media containing 28 g/L NaCl, 3.5 g/LMgSO₄.7H₂O, 2.7 g/L MgCl₂.6H₂O, 0.33 g/L KCl, 0.25 g/L NH₄Cl, 0.14 g/LCaCl₂.2H₂O, 2.00 mg/L FeCl₃, 0.05 mg/L H₃BO₃, 0.05 mg/L ZnCl₂, 0.03 mg/LCuCl₂.2H₂O, 0.05 mg/L MnCl₂.4H₂O, 0.05 mg/L (NH₄)₂MoO₄, 0.05 mg/LAlKSO₄.2H₂O, 0.05 mg/L CoCl₂.6H₂O, 0.05 mg/L NiCl₂.6H₂O, 3.30 mg/LNa₂WO₄.2H₂O, 5 g/L maltose and yeast extract, 0.5 μg/L riboflavin, and20 μM uracil or 4 mM agmatine as needed. After these ingredients aredissolved, the media was made anaerobic by the addition of 0.5 g/Lcysteine HCl, 0.5 g Na₂S (dissolved in 50 mL water). Following thereductant 1.0 g/L NaHCO₃ was added along with 1 mM potassium phosphatebuffer (from a 1 M or 1000× stock at pH 6.8). If needed, the pH of themedia was adjusted to 6.8 with HCl before degasing. Cultures wereinoculated to 1×10⁷ cells/mL and incubated at 98° C. until celldensities reached 1×10⁸ cells/mL. Cultures were then cooled at roomtemperature until the temperature reached 70 to 75° C. when they wereplaced in an incubator set to a temperature in the range of 65 to 75° C.for up to 32 hours. Cell densities were calculated from counting asample in a Hausser counting chamber.

P. furiosus cell paste was anaerobically resuspended in 50 mM Tris pH8.0+DNase 1 (3 mL buffer/g cell paste). The slurry was stirred for 30minutes in an anaerobic chamber, lysing the cells by osmotic shock. Thecrude extract was then centrifuges at 100,000×g for 1 hour. Theresulting supernatant (S-100) was diluted (with 50 mM Tris pH 8.0) andre-concentrated 3 times with a 3 kDa centrifugation filter. The washedand concentrated S-100 was sealed in a vial to maintain anaerobicity andstored at −80° C.

Growth of M. sedula for biochemical assays and product analysis. M.sedula (DSM 5348) was grown autotrophically as described in Example 3.

M. sedula cell paste was anaerobically resuspended in 50 mM Tris pH 8.0and Dnase 1 (2 mL buffer/g cell paste). The slurry was stirred for 1hour in an anaerobic chamber, lysing the cells by osmotic pressure. Thecrude extract was then centrifuges at 100,000×g for 1 hour. Theresulting supernatant (S-100) was sealed in a vial to maintain anaerobicconditions and stored at −80° C.

NADPH-dependent assays for the E2, E2+E3 and E1+E2+E3 reactions of SP1(FIG. 36). All reactions were carried out in sealed anaerobic cuvettesat 75° C. containing 100 mM MOPS pH 7.5 (measured at room temperature),5 mM MgCl₂, 5 mM DTT and the cell-free extract of P. furiosus (0.25mg/ml). After addition of NADPH, the relevant CoA derivative and othersubstrates (see below), NADPH oxidation was determined by the absorbanceat 340 nm and rates were calculated based on the difference before andafter the addition of the CoA substrate.

E2 assay. The added substrates were 1 mM NADPH and 1 mM Succinyl-CoA.Note that E3 does not utilize succinic semialdehyde, the product of thereaction.

E2+E3 assay. The added substrates were 1 mM NADPH and 1 mM Malonyl-CoA.In this case E3 does utilize the product, malonate semialdehyde, in aNADPH-dependent reaction.

E1+E2+E3 assay. The added substrates were 1 mM NADPH, 1 mM Acetyl-CoA, 1mM ATP and 10 mM NaHCO₃. The product, malonyl CoA, is then used by E2and the product of that reaction, malonate semialdehyde, is then used asa substrate for E3, both in NADPH-dependent reactions.

The growth of the strain PF505 before and after the temperature shiftfrom 98° C. to 75° C. are shown in FIG. 17. The Specific activities ofE1, E2 and E3 in cell-free extracts of PF506 after the temperature shiftfrom 98° C. to 75° C. are shown in Table 9.

TABLE 9 Specific activities of E1, E2 and E3 in cell-free extracts ofPF506 after the temperature shift from 98° C. to 75° C. Specificactivity: μmol NADPH oxidized/min/mg Enzymes E1 + E2 + E3 E2 + E3 E2Substrate Acetyl-CoA Malonyl-CoA Succinyl-CoA ΔPdaD 0 0 0  0 hr 0.030.05 0.03 16 hr 0.03 0.54 0.16 32 hr 0.07 0.28 0.11 48 hr 0.07 0.08 0.01Msed 0.02 0.08 0.08 Msed (literature)* 0.07 0.42 0.20 *Published valueassayed aerobically at 65° C.: Berg, I. A. et al. 2007. Science. 318,1782-1786)

The specific activities of E1, E2 and E3 in extracts of PF506 werecomparable to those measured in extracts of M. sedula and to literaturevalues reported by others after the P. furiosus cells were grown forapprox. 16 hours at 75° C. No activity was measured in cells grown at98° C.

NADPH-dependent assay for E9 of the SP2B subpathway (FIG. 36). Assayswere carried out in sealed anaerobic cuvettes at 75° C. containing 100mM MOPS pH 7.5 (measured at room temperature), 5 mM MgCl₂, 5 mM DTT, 1mM NADPH and the cell-free extract of P. furiosus (0.25 mg/ml). Afteraddition of 1 mM succinic semialdehyde, NADPH oxidation was determinedby the absorbance at 340 nm and rates were calculated based on thedifference before and after the addition of the succinic semialdehyde.

Growth of P. furiosus strain MW43 at 95° C. and temperature shift from65° C. to 90° C. for 18 hrs (FIG. 38). Cultures were shifted from 95° C.and were incubated for 18 hr before harvesting. The maximum activity andspecific activity for E9 is seen after the cultures are shifted to 70°C. (for 18 hr), with lower values at 65 and 75° C. The production ofactive E9 decreases dramatically at 80° C. and above.

E9 temperature profile and stability in cell-free extracts of P.furiosus strain MW43 (FIG. 39). The specific activity of E9 in P.furiosus strain MW43 (grown at 70° C.) is about 10-fold higher that thanmeasured in M. sedula. The highest E9 specific activity was measured inMW43 cells grown at 70° C. even though in cell extracts the maximumactivity was above 80° C. and the enzyme has a half-life of ˜30 min at90° C. It was concluded that P. furiosus cells should be temperatureshifted from 95-98° C. to 70° C. for 18 hrs to obtain the highestactivities of M. sedula enzymes.

Example 5

Determination of E1 and E2 Activities in P. furiosus Strain PF506, itsParent Strain ΔPdaD, in P. furiosus Strain MW56 and its Parent StrainCOM1, and in M. sedula

Phosphate Assay for E1 (FIG. 40). Pf extract was added to 0.1 mg/mL inbuffer containing 100 mM MOPS pH 7.5 (at room temperature), 5 mM MgCl₂,and 5 mM DTT. Added substrates were 10 mM NaHCO₃, 1 mM ATP, and 1 mMAcetyl-CoA. The sealed anaerobic vials were incubated at 75° C. and 20μL samples were taken out at 0, 2, and 4 minutes and added to a 96 wellplate. The samples were diluted with 180 μL of water before the additionof 30 μL of BioVision (Mountain View, Calif.) phosphate assay reagent.Absorbance at 650 nm was measured and rates were calculated based on thedifference between the—Acetyl-CoA control for each sample.

Specific activities of E1 and E2 in cell-free extracts of recombinantand parent P. furiosus strains and in M. sedula (Table 10). The E1 andE2 assays were carried out at 75° C. as described in FIGS. 16 and 23,respectively. Specific activities are expressed as nmol phosphatereleased and nmol NADPH oxidized/min/mg, respectively.

TABLE 10 E1: E2: Acetyl-CoA Malonyl-CoA Cell-extract P_(i) release NADPHoxidation COM 1 <5 <5 ΔPdaD <5 <5 MW56 93 ± 10 92 (n = 4) (n = 1) PF50674 ± 19 248 ± 123 (n = 6) (n = 11) Msed 206 ± 49  143 ± 60  (n = 3) (n =3)

The specific activities of E1 and E2 in P. furiosus strains PF506 andMW56 are comparable to those measured in Msed but are not detected inthe P. furiosus parent strains.

Example 6

Production of 3HP by Cell-Free Extracts of P. furiosus Strains PF506 andMW56

Identification and quantitation of 3-hydroxypropionate produced by theSP1 pathway in cell-free extracts of P. furiosus strain PF506 and strainMW56. Two approaches were used to produce 3HP: 1. Using malonyl CoA withNADPH or H₂/NADP as the electron donor catalyzed by enzymes E2+E3 (andSHI to activate H₂); and 2. Using acetyl CoA plus CO₂ (bicarbonate) withNADPH or H₂/NADP as the electron donor catalyzed by enzymes E1+E2+E3(and SHI to activate H₂).

Detection and quantitation of 3-hydroxypropionate (3HP). 3HP produced incell-free extracts of P. furiosus was derivatized by two reactions andeach derivative was identified and quantitated by different approaches.

HPLC: 2-Nitrophenylhydrazine derivatization. The 3HP-hydrazide wasprepared and extracted from mixtures with ether. The ether-extracted3HP-hydrazide was identified by ESI-MS analysis. The ether-extracted3HP-hydrazide was quantitated after separation by HPLC.GC-MS: per-O-trimethylsilylate derivatization. The 3HP-TMS derivativewas both identified and quantitated using GC-MS analysis.

Methods used to identify 3-HP in cell-free extracts of P. furiosus.Production of 3-HP from malonyl CoA by E2+E3 and from acetyl CoA byE1+E2+E3. To the Pf extract (0.25 mg/mL) in buffer containing 100 mMMOPS pH 7.5, 5 mM MgCl₂, and 5 mM DTT, was added 1-2 mM Malonyl-CoA (forE2+E3) or 10 mM NaHCO₃, 2 mM ATP and 1 mM Acetyl-CoA (for E1+E2+E3). Theelectron source was 2 mM NADPH or 0.5 mM NADP⁺ with 100% H₂ in theheadspace. Sealed anaerobic vials were incubated at 75° C. for up to 2hours.

GC-MS detection of 3-HP. A sample of the enzyme assay mixture was spikedwith 20 μg of inositol as an internal standard. For hydrolysis ofproteins, the samples were freeze-dried, then incubated in 2 M TFA at80° C. for 1 hour then dried under nitrogen. The samples were thenper-O-trimethylsilylated by treatment with Tri-Sil (Pierce) at 80° C.for 30 minutes. GC-MS analysis of the TMS derivatives was performed onan AT 7890n GC interfaced to a 5975C MSD, using a Grace EC-1 column (30m×0.25 mm). The exact mass of 3-HP-TMS is 162.

2-Nitrophenyl hydrazine derivatization of 3HP. The steps to derivatize3HP were as follows. 1) Add 100 μL sample of cell-free extract to 200 μLethanol. 2) Add 200 μL 20 mM 2-nitrophenyl hydrazine in 100 mMHCL/ethanol (1:1). 3) Add 200 μL 250 mM1-Ethyl-3-(3-Dimethylaminopropyl)-N′-ethylCarbodiimide hydrochloride(1-EDC.HCL) in 3% pyridine in ethanol (v/v). 4) Heat sample at 60° C.for 20 minutes. 5) Add 100 μL of 15% (W/V) KOH. 6) Heat again at 60° C.for 15 minutes. 7) Let sample cool and acidify with 50% HCL to pHbetween 4-6. 8) Analyze 10-50 μL aliquots on the HPLC.

Ether extraction of 3HP-Hydrazide. This was accomplished by thefollowing steps. 1) Add 1 mL 1 M KPO₄ Buffer, pH 7.0 to cooled 800 μLderivatized sample. 2) Add 1 mL of ether to sample and mix well. 3)Centrifuge 10 min 6,000 g to separate the phases. 4) Remove top etherlayer and transfer to a new tube. 5) Repeat steps 2-4. 6) Evaporate theether. 7) Suspend the dried sample in 200 μL methanol or 0.05% TFA. 8)Run 10-50 μL aliquots on the HPLC.

HPLC detection of 3-HP-Hydrazide. The column and run conditions were asfollows: column, Supelco LiChrosorb RP-8 (5 μm); solvent system, A 0.05%TFA, B 100% acetonitrile; gradient 0-20 min, 0-100% B, 20-22 min: 100%B; flow rate: 1 ml/min; temperature: 30° C.

ESI-MS detection of 3-HP-hydrazide. The derivatized 3HP samples wereextracted with ether, dried, and re-constituted in methanol. Theresulting samples were analyzed by direct injection on a Perkin-ElmerAPI 1 plus in negative mode. The exact mass of the anion 3HP-Hydrazideis 224.

Summary of methods used to identify 3-HP in cell-free extracts of P.furiosus is shown in Table 13. Summary of amounts of 3-HP produced bycell-free extracts of PF506 and MW56 using malonyl CoA (E2+E3) or acetylCoA+CO₂ (E1+E2+E3) as the carbon sources with NADPH or H₂ as theelectron donor is shown in Table 11 and Table 12

TABLE 11 E2 + E3: 1 mM E1 + E2 + E3: 1 mM Malonyl-CoA Acetyl-CoA NADPHNADPH e⁻ donor H₂ e⁻ donor e⁻ donor H₂ e⁻ donor Strain (2 mM) (100%headspace) (2 mM) (100% headspace) MW56 HPLC (not done) HPLC HPLC PF506HPLC GC-MS HPLC ESI-MS GC-MS ESI-MS ESI-MS

TABLE 12 E2 + E3: 1 mM Malonyl-CoA E1-E2 + E3: 1 mM Acetyl-CoA H₂ e⁻donor H₂ e⁻ donor P. furiosus NADPH e⁻ donor (100% NADPH e⁻ donor (100%strain (2 mM) headspace) (2 mM) headspace) MW0056 100 μM/30 min (notdone) 40 μM/8 min (D) 48 μM/8 min (D) (C) PF506 160 μM/2 hr (A) 150 μM/2hr 50 μM/2 min (D) 23 μM/2 min (D) 500 μM/2 hr (B) (A) 80 μM/30 min (C)

TABLE 13 A B C D Method GC-MS HPLC HPLC HPLC [Protein] 0.25 mg/mL 0.25mg/mL 0.3 mg/mL 3 mg/mL

Example 7

Production of 3HP by Whole Cells of P. furiosus Strains PF506 and MW56Product Analysis of E1+E2+E3 Activities in Whole Cells of P. furiosus.

In vivo 3-HP production assay. PF506 and MW56 were grown in 2 μLcultures at 98° C. for 10 hours until cell densities reached 1×10⁸cells/mL when they were cooled and incubated at 75° C. for 16 hours.Harvested cells were suspended to 5×10¹⁰ cells/mL in 100 mM MOPS pH 7.5and 1×Pf base salts (28 g/L NaCl, 3.5 g/L MgSO₄.7H₂O, 2.7 g/LMgCl₂.6H₂O, 0.33 g/L KCl, 0.25 g/L NH₄Cl, 0.14 g/L CaCl₂.2H₂O). The cellsuspension was sealed in a serum vial, degasses with Ar, and brought to0.5 g/L cysteine HCl. Added substrates were 10 mM NaHCO₃ and either 10mM maltose or 40 mM pyruvate. The vials were then degassed with H₂ andincubated at 75° C. for 60 minutes. Samples for 3-HP analysis by HPLCinclude a direct sample of the cell suspension, the supernatant of aportion, and the pellet re-suspended and lysed in water. A schematic ofhow P. furiosus metabolizes maltose and provides acetyl CoA for 3HPproduction is shown at FIG. 41.

A total of 135 μM of 3HP was produced by a cell suspension of MW56(5×10¹⁰ cells/ml) after 60 min at 75° C. A total of 199 μM of 3HP wasproduced by a cell suspension of PF506 (5×10¹⁰ cells/ml) after 60 min at75° C. 3-HP production by whole cells of P. furiosus strains PF506 andMW56 is summarized in Table 14. The majority (˜70%) of in vivo produced3-HP was contained within intact cells.

TABLE 14 1 mL cell MW56 PF506 suspension Pyruvate Maltose PyruvateMaltose No Substrate Extracellular  45 nmol 30 nmol <20 nmol  45 nmol<20 nmol Intracellular 110 nmol 80 nmol  50 nmol 100 nmol <20 nmol

Example 8 Anapleurosis and Assimilation of Acetyl-CoA Associated withH₂—CO₂ Autotrophy in the Thermoacidophilic Archaeon MetallosphaeraSedula

Metallosphaera sedula is an extremely thermoacidophilic archaeon(T_(opt)=73° C., pH 2.0) that grows heterotrophically on peptides andchemolithoautotrophically on metal sulfides or hydrogen gas (Auernik andKelly, 2010, Appl. Environ. Microbiol. 76:931-935). Forchemolithotrophic growth it uses a unique carbon fixation pathway, knownas the 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle (Berg etal., 2007. Science 318:1782-1786), so far found only in members of theorder Sulfolobales. This cycle is one of two such cycles foundexclusively in thermophilic archaea, the other being thedicarboxylate/4-hydroxybutyrate (DC/4HB) cycle present in the orderDesulfurococcales (Berg et al., 2010. Nat. Rev. Microbiol. 8:447-460).In the first part of the 3HP/4HB cycle, acetyl-CoA (C2) is convertedinto succinyl-CoA (C4) by two successive carboxylation steps (Berg etal., 2010. Nat. Rev. Microbiol. 8:447-460). Succinyl-CoA is thenconverted to 4HB, which is rearranged and cleaved to produce twomolecules of acetyl-CoA. Labeling studies using 4-hydroxy[1-¹⁴C]butyrateand [1,4-¹³C₁]succinate revealed how the 3HP/4HB pathway relates to M.sedula central metabolism (Estelmann et al., 2011. J. Bacteriol.193:1191-1200), suggesting that most of the carbon flux (abouttwo-thirds) enters central metabolism via succinate (‘succinate branch’)and not through reductive carboxylation of acetyl-CoA to pyruvate. Theremaining third of the carbon flux (‘acetyl-CoA branch’) passes through4HB, thereby regenerating acetyl-CoA (FIG. 42).

Transcriptomic analysis of M. sedula cells grown under strictlyautotrophic conditions (H₂, CO₂) presented here supports the premisethat most carbon is assimilated via succinate and provides additionalinsights into the connections between the carbon fixation and centralmetabolism. This analysis indicates that acetyl-CoA assimilation occursduring formation of citric acid cycle intermediates (e.g., citrate andmalate) and also during isoprenoid-based lipid biosynthesis (Koga andMorii, 2007. Microbiol. Mol. Biol. Rev. 71:97-120; Boucher et al., 2004.Molecular Microbiology 52:515-527). Thus, the six enzymes in theacetyl-CoA branch of the 3HP/4HB cycle, which catalyze the rearrangementof succinyl-CoA to acetoacetyl-CoA with subsequent cleavage toacetyl-CoA, are essential not only for CO₂ fixation but also foranaplerosis of acetyl-CoA.

Most of the individual enzymes of the 3HP/4HB cycle have now beencharacterized biochemically, including methylmalonyl-CoA mutase(Msed_0639) and epimerase (Msed_0638, Msed_2055) (Han et al., 2012.Appl. Environ. Microbiol. 78:6194-6202), and acyl-CoA synthetase(Msed_0406) that catalyzes the ligation of 4HB to CoA (Example 1) (Table1). Here, we report the biochemical characteristics of two more enzymesof the 3HP/4HB pathway—4-hydroxybutyryl-CoA dehydratase (4hbd)(Msed_1321) and β-ketothiolase (Th1) (Msed_0656). Furthermore, the finalpart of the 3HP/4HB cycle was re-constituted in vitro to produceacetyl-CoA from 4HB, demonstrating that these enzymes are likelyinvolved in the functioning cycle. Finally, biochemical andtranscriptomic information was used to examine the connection of the3HP/4HB cycle to central metabolism in M. sedula.

Materials and Methods

Growth of M. sedula in a gas-intensive bioreactor. M. sedula (DSMZ 5348)was grown aerobically on DSMZ medium 88 at pH 2.0 in a 70° C. shakingoil bath. For routine small cultures (30 ml), heterotrophically growncells were supplemented with 0.1% tryptone, while autotrophically growncells were grown with the addition of 50 ml gas mix to the headspace(80% H₂, 20% CO₂). Cell growth was scaled-up from 300 ml in sealed1-liter bottles to 2 liters in a stirred bench-top glass fermentor(Applikon), agitated at 250 rpm. Two separately regulated gas feeds wereused—one for H₂/CO₂ mixture and one for air. The flow rates were heldconstant for all conditions at 1 ml/min for the H₂/CO₂ gas mixtures(composition: varied) and 100 ml/min for air (composition: 78% N₂, 21%O₂, 0.03% CO₂). The gas mixture compositions were as follows:autotrophic carbon-rich (ACR)—80% H₂ and 20% CO₂; autotrophic carbonlimited (ACL)—80% H₂ and 20% N₂; and heterotrophic (HTR)—80% N₂ and 20%CO₂ (with 0.1% tryptone added to medium). Tandem bioreactors were runsimultaneously and started with the same inoculum to generate biologicalrepeats. Cells were harvested at mid-exponential phase by rapid coolingwith dry ice and ethanol and then centrifuged at 6000×g for 15 min at 4°C.

M. sedula oligonucleotide microarray transcriptional response analysis.A spotted whole-genome oligonucleotide microarray was used fortranscriptional analysis, as described in Example 1. Total RNA wasextracted and purified using an RNeasy kit (Qiagen), reverse-transcribedwith Superscript III (Invitrogen), re-purified, and labeled with eitherCy3 or Cy5 dye (GE Healthcare). Labeled cDNA was then hybridized to themicroarray slide (Corning) at 42° C. Slides were scanned on a GenePix4000B Microarray Scanner (Molecular Devices, Sunnyvale, Calif.) and rawintensities were quantitated using GenePix Pro version 6.0. Datanormalization and statistical analysis were performed using JMP Genomics5 (SAS, Cary, N.C.). In general, significant differential transcriptionwas defined to be relative changes in expression of ≧2-fold (where a log2 value of ±1 means a 2-fold change) having p values of ≧5.4 (Bonferronicorrection equivalent to a p value of 4.0×10⁻⁶ for this microarray).Microarray data are available through the NCBI Gene Expression Omnibus(GEO) under accession number GSE39944.

Heterologous expression of M. sedula genes in E. coli. Msed_0406,Msed_1321, Msed_0399, and Msed_0656 were amplified from genomic DNAusing primers from IDT Technologies (Coralville, Iowa). Msed_0406 wascloned into pET46-Ek/LIC with an N-terminal His₆ tag, as described inExample 1. Msed_1321 was cloned with an N-terminal His₆ tag into amodified pETA vector, into which the anaerobic hya promoter from E. colihad been inserted to allow for anaerobically-regulated expression (Sunet al., 2010. PLoS One 5:e10526). Msed_0399 was cloned into pET21bwithout a His-tag, and Msed_0656 was cloned into pCDF-Ek/LIC with anN-terminal His₆ tag. All four constructs were individually cloned intoNovaBlue GigaSingles E. coli competent cells and selected by growth onLB-agar supplemented with antibiotic. Sequences were confirmed by EtonBiosciences, Inc. (Durham, N.C.). Next, the plasmids were transformedinto E. coli Rosetta 2 (DE3) cells for protein expression. Rosettastrains containing pET46-0406, pET21b-0399, and pCDF-0656 were grown andexpressed aerobically at 37° C. for 16 h in Studier's auto-inducingmedium ZYM-5052 (Studier, 2005. Protein Expression and Purification41:207-234). Rosetta cells containing pETA-1321 plasmid were grown in a2 L Applikon bioreactor (37° C., 800 rpm, pH 6.7, 0.5 slpm air) inStudier's non-inducing medium ZYM-505 with 0.5 mM FeCl₃. Cells weregrown until dissolved oxygen reached ˜30% of the initial level, at whichpoint 50 mM glucose was added and the air feed was switched to N₂ toinduce anaerobic expression. The cells were grown for another threehours before harvest.

Enzyme purification and biochemical assays. Lysis of aerobicallyexpressed proteins began with harvesting cells and centrifuging at6000×g for 15 min at 4° C. Cell pellets were re-suspended in lysisbuffer (50 mM sodium phosphate, 100 mM NaCl, 0.1% Nonidet P-40, pH 8.0)and lysed with a French pressure cell (two passes at 18,000 psi). Thelysate was centrifuged at 25,000×g for 15 min at 4° C. to removeinsoluble material. Native E. coli proteins were removed byheat-treating the extract at 65° C. for 20 minutes. Nucleic acids wereprecipitated by addition of streptomycin sulfate (1% w/v), and thenlysate was centrifuged again at 25,000×g for 15 min at 4° C. to removeprecipitated nucleic acids and heat-labile proteins. The soluble,heat-treated cell-free extract was sterile-filtered (0.22 μm) beforechromatographic purification. Msed_1321 was lysed and purified in a Coyanaerobic chamber (95% N₂, 5% H₂). The cell pellet was re-suspended inlysis buffer (20 mM Tris, 20 mM NaCl, 3.5 mM DTT, 1 mg/ml lysozyme, pH8.0) and incubated for 30 min at 37° C., followed by heat-treatment at65° C. for 30 min. Streptomycin sulfate (1% w/v) was added and thelysate was centrifuged at 25,000×g for 15 min at 4° C. The soluble,heat-treated cell-free extract was sterile-filtered (0.22 μm) beforepurification by column chromatography.

4-hydroxybutyrate:CoA ligase (Msed_0406) and acetoacetyl-CoAβ-ketothiolase Msed_0656 These enzymes were purified using a 1 ml HiTrapnickel column (GE Healthcare). The soluble, heat-treated lysate wasloaded onto the column with binding buffer (50 mM sodium phosphate, 300mM NaCl, 20 mM imidazole, pH 7.4) and the his-tagged enzyme removed withelution buffer (50 mM sodium phosphate, 300 mM NaCl, 500 mM imidazole,pH 7.4). The elution fractions containing enzyme were collected,concentrated, and dialyzed into reaction buffer (100 mM MOPS, pH 7.5),and then either stored at 4° C. for immediate use or mixed with glycerolto 20% and stored at −20° C. For 4-hydroxybutyrate:CoA synthetase(Msed_0406), a discontinuous assay was used to measuresubstrate-dependent disappearance of CoA at 70° C. using5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), as described in Example 1.For acetoacetyl-CoA β-ketothiolase (Msed_0656), enzyme activity wasmeasured using the discontinuous DTNB assay to measuresubstrate-dependent disappearance of CoA at 70° C. The reaction buffercontained 20 mM MOPS, pH 7.5, 5 mM MgCl₂, 0.2 mM CoA, 0.3 mMacetoacetyl-CoA, and purified enzyme.

Crotonyl-CoA hydratase/(S)-3-Hydroxybutyryl-CoA dehydrogenase(Msed_0399). The enzyme was purified first using a Q-sepharose HiLoad16/10 anion exchange column (GE Healthcare). The protein was loaded ontothe column with 20 mM Tris, pH 8.0, and eluted with 20 mM Tris, 1 MNaCl, pH 8.0 using a linear elution gradient. Fractions containingMsed_0399 were confirmed with SDS-PAGE, collected and dialyzed intobuffer for size exclusion chromatography (50 mM potassium phosphate, 150mM NaCl, pH 7.0). The partially-purified protein was further separatedon a HiLoad 26/600 Superdex 200 PG column, and the elution fractionscontaining Msed_0399 were collected, concentrated, and dialyzed intoreaction buffer (100 mM MOPS, pH 7.5), and then either stored at 4° C.for immediate use or mixed with glycerol to 20% and stored at −20° C.Enzyme activity was measured spectrophotometrically at 70° C. byfollowing NAD⁺ reduction at 340 nm (extinction coeffienct at 340nm=3,400 M⁻¹ cm⁻¹) (Berg et al., 2007. Science 318:1782-1786). The assaymixture contained 20 mM MOPS (pH 7.0), 5 mM MgCl₂, 2 mM NAD⁺, 0.5 mMcrotonyl-CoA or (S)-3-hydroxybutyryl-CoA, and purified enzyme. Thereaction mixture was preheated for 2 min at 70° C. and the reactioninitiated by addition of substrate.

4-hydroxybutyryl-CoA dehydratase (Msed_1321). The enzyme was purifiedusing a 5 ml Bio-Scale Mini Profinity IMAC cartridge (Bio-Rad). Thesoluble, heat-treated lysate was loaded onto the column with bindingbuffer (50 mM Tris, 300 mM NaCl, 3.5 mM DTT, 20 mM imidazole, pH 8.0)and the his-tagged enzyme removed with elution buffer (50 mM Tris, 300mM NaCl, 3.5 mM DTT, 500 mM imidazole, pH 8.0). The fraction collectorwas positioned inside the anaerobic chamber, and fractions containingenzyme were collected, concentrated, and dialyzed into reaction buffer(100 mM potassium phosphate, pH 7.5, 1 mM DTT). The enzyme solution waseither stored at room temperature inside the anaerobic chamber forimmediate use or mixed with glycerol to 20% and stored at −20° C. in asealed vacuum dessicator. Enzyme activity was measured aerobically in acoupled spectrophotometric assay at 70° C. The assay mixture contained20 mM sodium phosphate, 5 mM MgCl₂, 2 mM 4HB, 2 mM ATP, 1 mM CoA, 2 mMNAD⁺, 1 mM DTT, 1 mg/ml purified Msed_0406, and 42 ng/ml Msed_0399. Thereaction mixture was pre-heated for 5 min at 70° C. to allowaccumulation of 4HB-CoA, and then initiated by addition of purifiedMsed_1321. For the oxygen-sensitivity assay, Msed_1321 was washed with100 mM potassium phosphate, pH 7.5, to remove any DTT from the reactionbuffer. An aliquot of Msed_1321 was exposed to air, vortexed well, andtested with the coupled assay at the specified time intervals. Duringthe intervening time, the enzyme and reaction buffer was stored at 4° C.The reaction buffer was made without ATP, CoA, or NAD⁺—these were keptat −20° C. and added to the reaction at time of use.

Analysis of in vitro acetyl-CoA production. Enzymatic production ofacetyl-CoA from 4HB was performed in vitro at 70° C. Acetyl-CoA synthase(ACS) from Pyrococcus furiosus (Glasemacher et al., 1997. Eur. J.Biochem. 244:561-567) (Pf-ACS) was used to form acetate from acetyl-CoA,and the resultant mixture was derivatized to form the phenacyl esterusing dibromoacetophenone (adapted from (Durst et al., 1975. Anal. Chem.47:1797-1801)) and assayed using reversed-phase HPLC (Waters). TheAdams' Lab at University of Georgia generously provided a recombinant E.coli strain containing Pf-ACS. Heat-treated, cell-free extract from thisstrain was used in the following assay.

The reaction mixture (100 μl) consisted of 100 mM sodium phosphate, pH7.9, 5 mM MgCl₂, 3 mM ATP, 3 mM CoA, 3 mM NAD⁺, 3 mM 4HB, 1 mM DTT, 3 mMADP, purified recombinant Msed_0406 (500 ng/l), Msed_1321 (50 ng/μl),Msed_0399 (50 ng/μl), and Msed_0656 (50 ng/μl). The reaction mixture wasincubated at 70° C. for 20 minutes, after which 10 μl of Pf-ACS extractwas added before incubating an additional 10 minutes at 95° C. Thesample was cooled for room temperature, acidified with 50% H₂SO₄ to pH2, and ether extracted twice with 750 ml diethyl ether. The etherfraction was neutralized with 50 μl 20 mM bicarbonate and dried down ina vacuum centrifuge for 2 hours at 30° C. The sample was resuspended in50 μl acetonitrile with 0.5 μl of 0.5% phenolphtalein. A solution of 100mM KOH was added until the sample turned pink (pH˜9-10), after which 100μl of acetonitrile, 50 μl of 1 μM 15-crown-5-ether, and 200 μl of 20 mM2,4-dibromoacetophenone were added. The solution was heated to 80° C.for 30 minutes, cooled back to room temperature, and injected (5 μl)onto a C18 silica-based column (Shodex C18-4E, 4.6×250 mm) at 30° C. Theinitial mobile phase composition was 60% Buffer A (0.05% trifluoroaceticacid) and 40% Buffer B (acetonitrile). Samples were eluted with a tenminute linear gradient to a final composition of 20% Buffer A and 80%Buffer B.

Results and Discussion

Conversion of 4-hydroxybutyrate to acetyl-CoA in the 3HP/4HB cycle. Thefinal four steps in the acetyl-CoA branch of the 3HP/4HB pathway (whichconverts 4HB to acetyl-CoA) are putatively catalyzed by 4HB:CoA ligase(Msed_0406), 4-hydroxybutyryl-CoA dehydratase (Msed_1321), crotonyl-CoAhydratase/(S)-3-hydroxybutyryl-CoA dehydrogenase (Msed_0399), andacetoacetyl-CoA β-ketothiolase (Msed_0656) (FIG. 42). In order toconfirm that these enzymes converted 4HB to acetyl-CoA, and to considerpossible rate-limiting steps, recombinant versions were produced,purified to homogeneity, and characterized biochemically (FIG. 42, Table15). For three of the enzymes, recombinant versions were readilyproduced. However, initial attempts to produce recombinant4-hydroxybutyryl-CoA dehydratase (Msed_1321) were unsuccessful, possiblybecause this enzyme is oxygen-sensitive, based on the oxygen sensitivityof a well-studied homolog from an anaerobic bacterium, Clostridiumaminobutyricum (Scherf et al., 1993. Eur. J. Biochem. 215:421-429; Mühet al., 1996. Biochemistry 35:11710-11718; Martins et al., 2004. Proc.Natl. Acad. Sci. U.S.A. 101:15645-15649). As such, both expression andpurification of Msed_1321 were conducted under anaerobic conditions: anexpression system based on the hya promoter from E. coli (Sun et al.,2010. PLoS One 5:e10526) was used, and recombinant cell lysis andprotein purification were carried out in an anaerobic chamber (95% N₂,5% H₂). This approach resulted in production of soluble, active enzyme.However, subsequent testing of Msed_1321 revealed it to be much lessoxygen sensitive than its clostridial counterpoint. With a half-life ofroughly 4 days, Msed_1321 proved to be surprisingly robust in thepresence of oxygen. This increased oxygen tolerance relative to the C.aminobutyricum enzyme could be an adaptive trait associated with theaerobic environments inhabited by M. sedula.

TABLE 15 Kinetic Properties of selected M. sedula enzymes from theacetyl-CoA branch of the 3HP/4HB pathway V_(max) (μmol min⁻¹ K_(m)k_(cat) k_(cal)/K_(m) Enzyme ORF mg⁻¹) (mM) (s⁻¹) (s⁻¹ M⁻¹) 4- Msed_04061.7 2.0 1.8 910 Hydroxybutyrate:CoA synthetase 4-Hydroxybutyryl-Msed_1321 2.2 0.15 2.1 1.4 × 10⁴ CoA dehydratase Crotonyl-CoA Msed_039920 0.07 19 2.6 × 10⁵ hydratase (C-CoA) (S)-3-Hydroxybutyryl- Msed_0399(3HB- 16 0.06 15 2.6 × 10⁵ CoA dehydrogenase CoA) Acetoacetyl-CoAMsed_0656 1400 0.18 1000 5.6 × 10⁶ β-ketothiolase

In a previous study, two separate genes (Msed_0406 and Msed_0394) werefound to encode acyl-CoA synthetases with activity on 4HB (8). BothMsed_0406 and Msed_0394 showed activity on a broad range of linear,unsubstituted organic acids (C2-C5). Although Msed_0406 catalyzed CoAligation at a faster rate than Msed_0394 for the range of substrateconcentrations examined (V_(max)−1.7 and 0.22 μmol min⁻¹ mg⁻¹,respectively), it is possible that both enzymes contributed to this invivo activity in M. sedula. The reaction rate for 4-hydroxybutyryl-CoAdehydratase (Msed_1321), the subsequent enzyme in the cycle, iscomparable to Msed_0406. Thus, these two steps could be rate-limitingbottlenecks for the acetyl-CoA branch of the 3HP/4HB pathway, sincetheir activities are approximately 10-fold lower than that for the twosteps catalyzed by Msed_0399 (20 and 16 μmol min⁻¹ mg⁻¹ for thehydration and dehydrogenase reactions, respectively), and Msed_0656(1400 μmol min⁻¹ mg⁻¹). The high K_(m) value for Msed_0406 (2.0 mM)stands out as being much higher than the K_(m) values for the rest ofthe enzymes (Msed_1321-0.15 mM, Msed_0399-0.07 mM, Msed_0656-0.18 mM),and suggests that post-transcriptional mechanisms impact substrate entryinto the Acetyl-CoA branch.

HPLC was used to confirm production of acetyl-CoA from 4HB using allfour enzymes. To detect organic acids using HPLC, samples weredervatized with 2,4-dibromoacetophenone (DBAP) and then run on areversed-phase C18 column. The reaction mixture containing 4HB and allthe necessary cofactors and enzymes was first incubated at 70° C.,followed by addition of Pf-ACS and a second incubation at 95° C. toconvert all the acetyl-CoA to acetate. The HPLC chromatograms for thereaction mixture, control, and standards (FIG. 43) confirm the in vitroconversion of 4HB to acetate using recombinant versions of theseenzymes.

Refined autotrophic growth conditions for M. sedula transcriptomicanalysis. For initial efforts focusing on M. sedula under autotrophicconditions, cultures were grown in sealed bottles in a shaking orbitalbath starting with a known headspace gas composition (Auernik and Kelly,2010, Appl. Environ. Microbiol. 76:931-935). Mass transfer of H₂, CO₂,and O₂ into the liquid medium was neither controlled nor enhanced and,thus, growth was subject to significant diffusional limitations. Toaddress this issue here, gas-intensive aerobic growth of M. sedula wasoptimized by controlling gas feed to a 3 L bioreactor. Gas feed rateswere controlled using rotameters and a micro-bubble sparging stone (2-μmpore size) was used to increase dissolution of sparingly soluble gases,H₂ in particular. The doubling time for M. sedula exponential growth forH₂—CO₂ autotrophy decreased from 11-13 h in sealed bottles to 5-6 hoursin the gas-intensive bioreactor, indicative of significant gas-liquidmass transfer limitations in the static cultures. These improveddoubling times for autotrophic growth were comparable to heterotrophicgrowth on 0.1% tryptone (5-6 hours), suggesting that metaboliclimitations from gas supply were alleviated to a significant extent.

Possible limitations of CO₂ gas-liquid mass transfer were investigatedfor three growth conditions: autotrophic carbon-rich (ACR) (80% H₂ and20% CO₂), autotrophic carbon-limited (ACL) (80% H₂ and 20% N₂), andheterotrophic (HTR) (80% N₂ and 20% CO₂) with 0.1% tryptone supplementedto the medium. In the ACL condition, all available inorganic carbon camefrom atmospheric CO₂ in the air feed. The observed growth rate for HTRand ACR cultures was comparable (t_(d)=6.7 h and 6.8 h, respectively),and faster than for the ACL culture (t_(d)=9.4 h).

Of the 2293 protein-coding genes in the M. sedula genome, 984 responded2-fold or more when comparing HTR with the ACL condition. While trendswere consistent with previous results from less defined growthconditions (Auernik and Kelly, 2010, Appl. Environ. Microbiol.76:931-935), in many cases, they were more pronounced. Among the mosthighly up-regulated genes for the HTR vs. ACL contrast were thosedirectly involved in CO₂ fixation in the 3HP/4HB pathway, especiallyacetyl-CoA/propionyl-CoA carboxylase (Msed_0147, Msed_0148, Msed_1375-7-to 30-fold), 4-hydroxybutyryl-CoA dehydratase (Msed_1321-27-fold), andcarbonic anhydrase (Msed_0390-29-fold). The effects of carbon dioxidelimitation was especially evident for the β-class carbonic anhydraseencoded in Msed_0390; transcript levels were induced 3.7-fold for thecarbon-rich (ACR) vs. heterotrophy (HTR) contrast, compared to nearly30-fold higher for the carbon-limited condition (ACL). This indicatesthat increasing the rate of bicarbonate formation from CO₂ is essentialfor rapid growth.

Bicarbonate formation actually depends on two separate reversibleequilibria—first, the hydration of aqueous CO₂ to form carbonic acid(H₂CO₃) and second, the first ionization of polyprotic carbonic acid toform bicarbonate (HCO₃ ⁻). In aqueous solution at 70° C., the reactionrate of the hydration step will be roughly 75-fold faster than at 25° C.(Wang et al., 2009. The Journal of Physical Chemistry A 114:1734-1740).The equilibrium constant only increases by 2-fold (2.4e-05 M⁻¹ at 25° C.vs 4.0e-05 M⁻¹ at 70° C.), so even at elevated temperatures theconcentration of aqueous CO2 is roughly 25,000 times greater than theconcentration of carbonic acid (Wang et al., 2009. The Journal ofPhysical Chemistry A 114:1734-1740). However the apparent pKa of theionization of carbonic acid to bicarbonate at 65 C is about 6.3, whichmeans that at low pH virtually no bicarbonate exists in solution. M.sedula therefore likely uses carbonic anhydrase to convert CO₂ tocarbonic acid in the cytoplasm, where the pH value is closer to neutral.The intracellular pH of Sulfolobus acidocaldarius, an acidophilicarchaeon very closely related to M. sedula, has been measured to bearound 6.5 (Baker-Austin et al., 2007. Trends Microbiol. 15:165-171). Atthat pH, the rapid ionization of carbonic acid to bicarbonate wouldprovide the necessary substrate for carbon fixation and cellular growth.

The transcriptional response data from growth under gas-intensiveconditions provided a clearer picture of the role of genes associatedwith M. sedula hydrogenases in H₂—CO₂ autotrophy. Genes encoded inMsed_0913-0950 were all up-regulated. This locus encodes the two Ni—Fehydrogenases (Msed_0923-0924, Msed_0944-0945), multiple accessoryproteins (HypABCDF), a maturation protease (Msed_0916), and additionalhypothetical proteins that were all highly up-regulated underautotrophy. The only potential hydrogenase-related protein notassociated with this locus is Msed_2256, which has 48% amino acididentity over the entire open reading frame to SlyD from Pyrococcusfuriosus, a chaperone protein that participates in the recruitment ofHypB, a nickel-binding GTPase (Chung et al., 2010. FEBS Lett.585:291-294). Msed_2256 was not up-regulated under H₂—CO₂ autotrophy,but is constitutively transcribed at high levels under all growthconditions. Transcripts for both Ni—Fe hydrogenases were up-regulated 5to 10-fold under autotrophy, although their absolute transcript levelsdiffered significantly; Msed_0944-0945 was transcribed at ˜30-foldhigher levels than Msed_0923-0924 for both heterotrophy and autotrophy.Msed_0943-0950, which encodes a membrane-associated hydrogenase, wasstrongly up-regulated under autotrophic conditions (Msed_0949—48-foldincrease; Msed_0948—42-fold increase; Msed_0947—19-fold increase). ThisNi—Fe hydrogenase is likely the primary enzyme responsible for energyconservation via molecular hydrogen oxidation.

Assimilation and anapleurosis of acetyl-CoA during H₂—CO₂ autotrophy.Taken together, the transcriptomics data acquired through thegas-intensive bioreactor provided a more complete perspective on theassimilation and anapleurosis of acetyl-CoA during growth of M. sedulaby H₂—CO₂ autotrophy. FIG. 44 summarizes these data for genes implicatedin CO₂ fixation and central metabolism (adapted from (Estelmann et al.,2011. J. Bacteriol. 193:1191-1200)). Acetyl-CoA is shown in red boxes tohighlight where it is produced or required. The schematic includes theinitial steps of the isoprenoid-based lipid biosynthesis pathway(mevalonate pathway) and amino acid biosynthesis groups (shown in blackboxes). Note that both PEP carboxylase and PEP carboxykinase areincluded in FIG. 44 (Enzymes 27 and 28). Assays of M. sedula extractdetected activity for PEP carboxykinase (70 nmol min⁻¹ mg⁻¹ inautotrophic extracts), but no activity for PEP carboxylase in eitherautotrophic or heterotrophic extracts was found (Estelmann et al., 2011.J. Bacteriol. 193:1191-1200).

The transcription of most genes directly involved in the 3HP/4HB CO₂fixation cycle (see upper right in FIG. 44) were triggered underlimiting CO₂ concentrations. However, the genes encoding the incompletetricarboxylic acid cycle (TCA) are not as responsive, suggesting othermechanisms of regulation. These data support previous carbon fluxanalysis for the 3HP/4HB pathway that showed that carbon from CO₂ enterscentral metabolism via succinate (Estelmann et al., 2011. J. Bacteriol.193:1191-1200). Genes encoding succinate dehydrogenase (Msed_0674-0677)were constitutively transcribed at high levels (75% percentile of thetranscriptome), along with a gene annotated as fumarate hydratase(Msed_1462) (70% percentile). No strong transcriptional response wasobserved for potential candidates for succinic semialdehydedehydrogenase (Msed_0367, Msed_1298, or Msed_1774) or succinyl-CoAsynthetase (Msed_1581-1582); transcripts for Msed_1581-1582, wereactually down-regulated under autotrophy (6-fold and 5-fold,respectively). This is consistent with the low activity measured forsuccinyl-CoA synthase in extracts from heterotrophically grown cells(146 nmol min⁻¹ mg⁻¹), which was actually higher than the activity inextracts from autotrophically grown cells (36 nmol min¹ mg⁻¹).Transcript levels for possible succinic semialdehyde dehydrogenasecandidates varied: Msed_1774 decreased under autotrophy (down 3.6-fold),while Msed_0367 and Msed_1298 showed no differential response andaverage transcript levels relative to the transcriptome. Whether theseORFs have been correctly annotated or whether other unidentified genesare responsible for these biotransformations remains to be seen.

Metabolic flux analysis of M. sedula metabolism using labeled4-hydroxy[1-¹⁴C]butyrate and [1,4-¹³C₁]succinate showed an unexpectedroute linking the carbon fixation cycle to central metabolism (Estelmannet al., 2011. J. Bacteriol. 193:1191-1200). Initially, it was suggestedthat acetyl-CoA was reductively carboxylated directly to pyruvate bypyruvate synthase (Berg et al., 2007. Science 318:1782-1786). However,the labeling patterns of the amino acids did not support thishypothesis, and instead it was argued that the major flux from thecarbon fixation pathway happens via succinyl-CoA. Oxidation ofsuccinyl-CoA to malate and oxaloacetate yield pyruvate andphosphoenolpyruvate (PEP), respectively. Therefore, to make one moleculeof pyruvate with the 3HP/4HB pathway, it requires 1.5 turns of thecycle—one full turn to make acetyl-CoA and another half-turn to makesuccinyl-CoA. In the anaerobic DC/4HB pathway, pyruvate can be formeddirectly from acetyl-CoA by reductive carboxylation. This makes theaerobic 3HP/4HB pathway nearly twice as expensive energetically,requiring nine ATP equivalents to make one molecule of pyruvate comparedto five for the DC/4HB pathway (Berg et al., 2010. Nat. Rev. Microbiol.8:447-460; Estelmann et al., 2011. J. Bacteriol. 193:1191-1200).

Although the genes encoding for succinic semialdehyde dehydrogenase orsuccinyl-CoA synthetase were not transcriptionally responsive, the datado not preclude their involvement. In the case of succinic semialdehydedehydrogenase, it may be that there are as yet unidentified genesresponsible for the conversion. Clearly the activity levels aresufficient for the transformations, and the labeling data unambiguouslysupports the primacy of the succinate branch for carbon flux intocentral metabolism.

Beyond succinyl-CoA, it appears that acetyl-CoA assimilation still hasan important role as a biosynthetic precursor based on the total cellcarbon measured in the labeling studies (Estelmann et al., 2011. J.Bacteriol. 193:1191-1200). This does not occur through reductivecarboxylation of acetyl-CoA to pyruvate, but instead throughincorporation into other central carbon intermediates, such as citrateand malate (FIG. 44). Acetyl-CoA is also essential for isoprenoid-basedlipid biosynthesis in Archaea (Koga and Morii, 2007. Microbiol. Mol.Biol. Rev. 71:97-120; Boucher et al., 2004. Molecular Microbiology52:515-527), and indeed, 33% of the 4-hydroxy[1-¹⁴C]butryate label fedto autotrophically growing M. sedula ended up in the lipid and pigmentfraction (Estelmann et al., 2011. J. Bacteriol. 193:1191-1200). Theinitial steps of isoprenoid biosynthesis require the condensation of twomolecules of acetyl-CoA to acetoacetyl-CoA (FIG. 42). When growingautotrophically, acetoacetyl-CoA could be recruited directly from the3HP/4HB pathway, or alternatively formed by acetyl-CoA acetyltransferase(Msed_1647). 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) is formed byHMG-CoA synthase (Msed_1646) and reduced to mevalonic acid (Matsumi etal., 2011. Res. Microbiol. 162:39-52). Interestingly, both Msed_1646 andMsed_1647 are expressed 5-fold higher under HTR compared to ACL.However, HMG-CoA is also produced during the catabolism of leucine,which may account for the increased expression levels of these twogenes. The remaining enzyme in the synthesis of mevalonate (Msed_1649)did not show any differential transcription between the tested growthconditions.

The assimilation of acetyl-CoA directly into central carbonintermediates occurs both through citrate and malate synthesis. Thereare two genes in M. sedula annotated as citrate synthase, Msed_0281 andMsed_1522, which under autotrophy were down-regulated 7-fold andup-regulated 3.8-fold, respectively. The role of malate synthase in M.sedula metabolism is still uncertain. There is a gene in the M. sedulagenome annotated as malate synthase, Msed_1042, that is constitutivelyexpressed at high levels (80% percentile) and malate synthase activityhas been measured in autotrophic cell extracts (58 nmol min⁻¹ mg⁻¹)(Estelmann et al., 2011. J. Bacteriol. 193:1191-1200). However, M.sedula does not have a gene for isocitrate lyase and activity was foundneither in autotrophic nor heterotrophic extracts, which suggests thatglyoxylate is not being formed from isocitrate to prevent loss of CO₂.Thus it is unclear what role malate synthase has in M. sedula, or howthe glyoxylate is being formed. The recent report of malate synthaseparticipation in pentose metabolism in Sulfolobales does not appear tobe related, as M. sedula does not grow on sugars (Nunn et al., 2010. J.Biol. Chem. 285:33701-33709).

Regulation of flux between the succinate and acetyl-CoA branches.Succinyl-CoA, therefore, represents a branching point where carbon fluxeither proceeds towards malate or continues through the cycle to 4HB andacetyl-CoA. The enzymes utilized in the acetyl-CoA branch wereexpressed, biochemically characterized, and the sub-pathwayreconstructed in vitro. Based on enzyme kinetic data, flux through theacetyl-CoA branch appears dependent on the activity of 4-HB-CoAsynthetase. Previous work has established that activity of acetyl-CoAsynthetase is controlled by acetylation of a conserved lysine residue bySir2 (Starai et al., 2002. Science 298:2390-2392). Regulation on 4HB-CoAsynthetase makes sense from an energetic standpoint, since this reactionrequires an investment of 2 ATP equivalents to activate 4HB and form thethioester bond. Thermodynamically, this investment is not essential forthe transformation of 4HB to acetate, but the formation of thehigh-energy thioester bond serves to help overcome other, lessthermodynamically favorable reactions elsewhere in the carbon fixationpathway, such as carboxylation and carbonyl reduction reactions(Bar-Even et al., 2012. Biochim. Biophys. Acta 1817:1646-1659). The highMichaelis-Menten constant for Msed_0406 (2 mM) indicates thatintracellular levels of 4HB must be high to overcome the activitybarrier. The reaction rate for the subsequent transformation, thedehydration by 4HBD, is also slow (2.2 μmol min⁻¹ mg⁻¹) and, hence,these two reactions form the rate-limiting steps for the acetyl-CoAbranch. The final three reactions, catalyzed by the bifunctionalcrotonyl-CoA hydratase/(S)-3-hydroxybutyrate dehydrogenase andacetoacetyl-CoA β-ketothiolase, have much faster reaction rates, 20/16μmolmin⁻¹ mg⁻¹ and 1400 μmolmin⁻¹ mg⁻¹, respectively. Taken together,these indicate that 4HB-CoA synthetase activity serves as the entrypoint both kinetically and energetically to the acetyl-CoA branch, andas such is the primary determinant of carbon flux distribution.

Oxygen tolerance of 4-hydroxybutyryl-CoA dehydratase from M. sedula.Here, we also report the cloning and characterization of4-hydroxybutyryl-CoA dehydratase (4hbd-Msed_1321), the first recombinanthomolog of this unique enzyme cloned from an archaeal host. Firstdiscovered in Clostridium aminobutryicum (Gerhardt et al., 2000. Arch.Microbiol. 174:189-199), this enzyme has drawn a lot of interest due toits unusual radical-based catalysis mechanism (Martins et al., 2004.Proc. Natl. Acad. Sci. U.S.A. 101:15645-15649; Buckel et al., 2006.Annu. Rev. Microbiol. 60:27-49). The clostridial verison is active as ahomotetramer with one [4Fe-4S]²⁺ cluster and one flavin adeninedinucleotide (FAD) cofactor per subunit (Martins et al., 2004. Proc.Natl. Acad. Sci. U.S.A. 101:15645-15649). The M. sedula homolog was alsofound to associate as a homotetramer (222 kDa, subunit mass=56.6 kDa).When exposed to air, the clostridal homolog was slightly activated dueto oxidation of the FAD cofactor, followed by oxidation of the Fe—Scluster leading to irreversible inactivation in ˜25 min (Scherf et al.,1993. Eur. J. Biochem. 215:421-429). However, the M. sedula homolog wasmuch more robust to oxygen exposure. The same initial activation uponexposure to air was observed but lasted for about 15 hours instead ofmere minutes; enzyme activity continued to drop slowly over the courseof the next week, with an observed half-life of 4 days. This substantialincrease in oxygen tolerance is probably an adaptive trait acquired as aresult of the aerobic environment in M. sedula.

Transcriptional patterns of 2-oxoacid oxidoreductases in M. sedula.There are several operons in the M. sedula genome containing genesannotated as putative 2-oxoacid oxidoreductases, however the exactnature of their role in metabolism remains uncertain. Previous assayshave found very low levels of 2-oxoglutarate and pyruvate oxidoreductaseactivity in both heterotrophic and autotrophic cell extract (2-3 μmolmin⁻¹ mg⁻¹) (Estelmann et al., 2011. J. Bacteriol. 193:1191-1200). FIG.45 shows the transcriptional profile of putative 2-oxoacidoxidoreductases under heterotrophy and autotrophy. Most of these locieither showed no transcriptional change (Msed_0507-0510, Msed_1199-1201)or were down-regulated under autotrophy (Msed_0306-0309, Msed_0524-0525,Msed_1596-1597), however the most striking change was thedown-regulation of Msed_1596 and Msed_1597, whose transcription levelsdecreased 23- and 10-fold, respectively. This strong transcriptionalresponse clearly implicates Msed_1596-1597 in some crucial, but as yetunknown, role under heterotrophic growth.

Conclusion. Here, the recombinant expression and characterization of twoenzymes in the final steps of the 3HP/4HB pathway are reported and invitro production of acetate from 4HB is confirmed. The level ofbiochemical detail of the 3HP/4HB pathway in relationship to centralmetabolism continues to develop, which will inform future metabolicengineering prospects for microbial biosynthesis of fuels and organicchemicals (Hawkins et al., 2013. Curr. Opin. Biotechnol. 24:376-384).

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference in their entirety.Supplementary materials referenced in publications (such assupplementary tables, supplementary figures, supplementary materials andmethods, and/or supplementary experimental data) are likewiseincorporated by reference in their entirety. In the event that anyinconsistency exists between the disclosure of the present applicationand the disclosure(s) of any document incorporated herein by reference,the disclosure of the present application shall govern. The foregoingdetailed description and examples have been given for clarity ofunderstanding only. No unnecessary limitations are to be understoodtherefrom. The invention is not limited to the exact details shown anddescribed, for variations obvious to one skilled in the art will beincluded within the invention defined by the claims.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless otherwise indicated to thecontrary, the numerical parameters set forth in the specification andclaims are approximations that may vary depending upon the desiredproperties sought to be obtained by the present invention. At the veryleast, and not as an attempt to limit the doctrine of equivalents to thescope of the claims, each numerical parameter should at least beconstrued in light of the number of reported significant digits and byapplying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. All numerical values, however, inherently contain a rangenecessarily resulting from the standard deviation found in theirrespective testing measurements.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

1-2. (canceled)
 3. A genetically engineered microbe modified to convertacetyl CoA, molecular hydrogen and carbon dioxide to 4-hydroxybutyrate,wherein the 4-hydroxybutyrate is produced at increased levels comparedto a control microbe, wherein the microbe is a hyperthermophile, andwherein the microbe comprises an exogenous coding region encoding apolypeptide, wherein the polypeptide has an activity selected from3-hydroxypropionate:CoA ligase activity, 3-hydroxypropionyl-CoAdehydratase activity, acryloyl-CoA reductase activity, methylmalonyl-CoAepimerase activity, methylmalonyl-CoA mutase activity, and succinatesemialdehyde reductase activity. 4-6. (canceled)
 7. The geneticallyengineered microbe of claim 3 wherein the hyperthermophile is anarcheon.
 8. The genetically engineered microbe of claim 7 wherein thearcheon is a member of the Order Thermococcales, a member of the OrderSulfolobales, or a member of the Order Thermotogales.
 9. The geneticallyengineered microbe of claim 8 wherein the archeon is Thermococcuskodakarensis, T. onnurineus, Sulfolobus solfataricus, S. islandicus, S.acidocaldarius, or Pyrococcus furiosus.
 10. (canceled)
 11. Thegenetically engineered microbe of claim 3 wherein the microbe comprisesan exogenous coding region encoding a polypeptide, wherein thepolypeptide has an activity selected from acetyl/propionyl-CoAcarboxylase activity, malonyl/succinyl-CoA reductase activity, andmalonate semialdehyde reductase activity. 12-14. (canceled)
 15. Thegenetically engineered microbe of claim 3 wherein the microbe produces4-hydroxybutyrate, and wherein the microbe comprises an exogenous codingregion encoding a polypeptide, wherein the polypeptide has an activityselected from 4-hydroxybutyrate:CoA ligase activity, 4-hydroxybutyrl-CoAdehydratase activity, crotonyl-CoA hydratase/(S)-3-hydroxybutyrl-CoAdehydrogenase activity, and acetoacetyl-CoA β-ketothiolase activity. 16.(canceled)
 17. The genetically engineered microbe of claim 3 wherein anexogenous coding region is operably linked to a temperature sensitivepromoter, to a constitutive promoter, or to a non-regulated promoter.18. The genetically engineered microbe of claim 3 wherein the microbefurther comprises a hydrogenase.
 19. The genetically engineered microbeof claim 18 wherein the hydrogenase is a NADPH-dependent hydrogenase.20. The genetically engineered microbe of claim 19 wherein the microbecomprises exogenous coding regions encoding subunits of theNADPH-dependent hydrogenase.
 21. The genetically engineered microbe ofclaim 20 wherein the subunits of the NADPH-dependent hydrogenasecomprise a hydrogenase alpha subunit and a hydrogenase delta subunit.22. The genetically engineered microbe of claim 21 wherein the subunitsof the NADPH-dependent hydrogenase further comprise a hydrogenase betasubunit and a hydrogenase gamma subunit. 23-24. (canceled)
 25. A methodcomprising incubating the genetically engineered microbe of claim 3under anaerobic conditions suitable for converting acetyl CoA, molecularhydrogen, and carbon dioxide to 4-hydroxybutyrate.
 26. The method ofclaim 25 further comprising recovering the 4-hydroxybutyrate. 27-29.(canceled)
 30. The method of claim 25 wherein the incubating comprisesan incubation temperature of at least 75° C. 31-33. (canceled)
 34. Acell free composition that converts acetyl CoA, molecular hydrogen andcarbon dioxide to 4-hydroxybutyrate, wherein the composition comprises apolypeptide having 3-hydroxypropionate:CoA ligase activity, apolypeptide having 3-hydroxypropionyl-CoA dehydratase activity, apolypeptide having acryloyl-CoA reductase activity, a polypeptide havingmethylmalonyl-CoA epimerase activity, a polypeptide havingmethylmalonyl-CoA mutase activity, and a polypeptide having succinatesemialdehyde reductase activity.
 35. A cell free method for fixing CO₂comprising incubating the cell free composition of claim 34 underanaerobic conditions suitable for the fixation of CO₂ by the conversionof acetyl CoA, molecular hydrogen and carbon dioxide to4-hydroxybutyrate.
 36. The cell free method of claim 35 furthercomprising isolating the 4-hydroxybutyrate. 37-40. (canceled)
 41. Thecell free composition of claim 34 wherein the cell free compositionfurther comprises a polypeptide comprising acetyl/propionyl-CoAcarboxylase activity, a polypeptide comprising malonyl/succinyl-CoAreductase activity, a polypeptide comprising malonate semialdehydereductase activity, and a polypeptide comprising NADPH-dependenthydrogenase activity.